Coagulation / transfusion medicine Flashcards
Clotting factors in blood and their synonyms
Define thrombophilia
o AKA hypercoagulability
o Describes a propensity for inappropriate thrombus formation.
T/F Many of the factors that reduce clot formation are activated by the products of procoagulant factors.
TRUE
How many causes of inherited hypercoagubility have been described in veterinary medicine?
o Thrombophilia is a result of inherited or acquired causes.
o No inherited forms of thrombophilia have been described in veterinary medicine.
How many causes of inherited hypercoagubility have been described in veterinary medicine?
o Thrombophilia is a result of inherited or acquired causes.
o No inherited forms of thrombophilia have been described in veterinary medicine.
Virchow’s triad
o Edothelial dysfunction
o Hypercoagulability
o Blood stasis or altered blood flow
o In most clinical scenarios, these contributors overlap. For instance, endothelial dysfunction leads to numerous alterations (e.g., loss of thrombomodulin function, release of von Willebrand multimers) that ultimately affect the coagulability of blood.
T/F Widespread coagulation perpetuates the inflammatory response by direct activation of inflammatory mediators
TRUE
T/F Thrombin can induce directly inflammatory cytokine production, and microthrombosis, which leads to tissue hypoxia and possible reperfusion injury
TRUE
What are the main mechanism of hypercoagulability? (categories)
o Endothelial disturbances
o Increased procoagulant elements
o Decreased endogenous anticoagulants
o Perturbations in fibrinolysis
Components of the endothelial barrier
o Vascular endothelial cells
o Glycocalyx
Composition of glycocalyx
o The glycocalyx comprises a large network of negatively charged glycosaminoglycans (GAGs), proteoglycans, and glycoproteins.
o Heparan sulfate accounts for 50% to 90% of the proteoglycans and facilitates the binding of antithrombin, which increases the efficiency of AT-mediated inhibition of thrombin.
o Other important anticoagulants bind the glycocalyx, including heparin cofactor II and TM.
o Tissue factor pathway inhibitor (TFPI) localizes to the glycocalyx, occurring either via heparan sulfate or via a glycosylphosphatidylinositol - lipid anchor.
o The glycocalyx also serves as a mechanoreceptor, sensing altered blood flow and releasing nitric oxide during conditions of increased shear stress to maintain appropriate organ perfusion.
o Nitric oxide (NO) has important effects on the inflammatory response, leukocyte adhesion to the endothelium, and inhibition of platelet aggregation.
How can inflammation affect glycocalyx?
o With inflammation, synthesis of the GAGs is decreased
o Therefore, the function of key anticoagulants that rely on the glycocalyx (e.g., TM and protein C, TFPI) is decreased.
o The glycocalyx also buffers endothelial cells by preventing the binding of inflammatory cytokines to cell surface receptors.
By which molecules can the endothelial cells be activated?
o Tumor necrosis factor-α (TNF-α)
o Bradykinin
o Thrombin
o Histamine
o Vascular endothelial growth factor (VEGF)
What does the Weibel Palade bodies contain?
o vWF
o Tissue plasminogen activator (tPA)
o P-selectin
o IL8
o Factor VIII
Once endothelial cells are activated, what will they release?
o Ultralarge multimers of vWF from Weibel-Palade bodies
What will the ultra large multimers of vWF do once released by the activated endothelial cells?
o They will bind platelet receptor GP Ib alpha, initiating PLT activation
T/F Small multimers of vWF are more active for PLT adhesion and activation than large multimers of vWF
FALSE - UL-vWF are more active than smaller multimers
What happens in health when UL-vWF are released?
o They are quickly cleaved into smaller multimers by a disintegrin-like and metalloproteinase with thrombospondin type 1 repeats (ADAMTS13).
o These smaller vWF molecules circulate freely in association with FVIII and have considerably less platelet aggregatory activity than the UL-vWF molecules.
o The UL-vWFs usually remain tethered at sites of endothelial activation or injury, bound to the cell surface or to exposed collagen.
What happens with decreased levels of ADAMTS13?
o A decrease or absence of ADAMTS13 may result in high concentrations of UL-vWF, which then can cause systemic platelet aggregation, thrombosis, and a subsequent consumptive thrombocytopenia (thrombotic thrombocytopenic purpura, reported in people)
o Acquired TTP has been reported in human patients who have developed antibodies against ADAMTS13 and in patients exposed to certain drugs such as clopidogrel or cyclosporine. Patients with certain malignancies and systemic lupus erythematosus are also at risk.
o Lower ADAMTS13 levels resulting from inflammatory disease may contribute to pathologies seen with other coagulopathies (DIC).
The activated endothelium will release the Weibel Palade content with ULvWF. What else will be exposed with endothelial activation / disruption and what are the consequences?
o Tissue factor
o TF will bind to circulating FVII and start coagulation
o TF it is expressed on surface of activated monocytes/macrophages and neoplastic cells
o Will perpetuate inflammation by activating nuclear factor kappa beta (NFkB) that will stimulate the production of TNFalpha
Platelets are activated when ULvWF multimers bind to their GP Ib alpha receptors. What will happen with those platelets upon activation?
o They change in shape
o They shuffle from the inside to the outside phosphatidylserine and phosphatidylethonalamine, negatively charged phospholipids (they will be the catalytic surface for tenase and prothrombinase complexes for the propagation phase of clot formation).
o They increase the expression of fibrinogen receptors -> GP IIb IIIa (aka intern alpha2b, B3)
o They release the content of their alpha and dense granules
What are microparticles?
o Microparticles (MPs) are circulating small vesicles (membrane blebs) released from activated or apoptotic cells.
o MPs may be derived from platelets, ECs, leukocytes, erythrocytes, and neoplastic cells.
What is the role of Microparticles in coagulation?
o Like platelets, MPs also can provide an asymmetric phospholipid membrane for thrombin generation.
o MPs can express TF on their surface, and those expressing phosphatidylserine and TF are characterized as procoagulant MPs.
o TF-bearing MPs originating from granulocytes and platelets have been identified in people with sepsis.
o TF-bearing MPs have been shown to induce coagulation in vitro through the VIIa-TF pathway.
o Some evidence suggests the presence of increased circulating TF activity in dogs with IMHA, which may be a result of TF-bearing MPs.
o Other procoagulant MPs may display vWF-binding sites and UL-vWF multimers, which can tether and activate circulating platelets.
T/F Anticoagulant factors are released once pro coagulation and clot formation is finished
FALSE - The nearly simultaneous activation of anticoagulant factors, even while clot propagation is still occurring, helps to prevent a procoagulant state or the systemic dissemination of coagulation.
What are the main anticoagulant proteins?
o Antithrombin
o Protein C
o Tissue Factor Pathway Inhibitor
T/F Many anticoagulant factors exist, with an anticoagulant described for nearly every procoagulant element.
TRUE
T/F - AT, TFPI, and the protein C system are only anticoagulant molecules
FALSE - they are directly or indirectly antiinflammatory.
What are the functions / effects of antithrombin?
o Inhibits thrombin formation
o Inhibits FXa
o Less inhibitory effects on FIXa and FVIIa-TF complex
o Most effective when bind to heparin-like GAGs (heparan sulfate) or when exposed to exogenous heparins (thrombin inhibition increased x 1,000)
o In absence of heparins and in presence of thrombin -> AT + TM will bind and inhibit thrombin.
In which situations will antithrombin be decreased?
o Systemic inflammation or critical illness: consumption (because of thrombin generation), decreased production (negative acute phase protein), or degradation by neutrophil elastase.
o Urinary loss of AT also may occur in animals with glomerulonephritis.
Protein C system in health
o Inhibits FVa and FVIIIa -> inhibition accelerated x20 when cofactor protein S is present
o Activated when TM binds thrombin on endothelium, mainly of microcirculation -> becomes APC
o Endothelial protein C receptor (EPCR) accelerates thrombomodulin binding thrombin
o TM + thrombin has 3 main functions:
* Helps generate APC
* Prevents thrombin from acting on fibrinogen and PLT
* Will generate TAFI - thrombin activatable fibrinolysis inhibitor -> inhibits fibrinolysis
Protein C system during inflammation
o Decreased hepatic production of protein C and protein S
o Decrease activation of protein C due to the effects of inflammatory cytokines on the endothelium and thrombomodulin.
o TNF alpha downregulates the expression of TM
o Elastase (produced by endotoxin activated neutrophils) cleaves TM from endothelium
o Circulating or soluble TM less effective than when complexed with EPCR on endothelium.
o Soluble TM is increased in people with sepsis and independently predicts the presence of DIC, MODS, and mortality.
Tissue Factor Pathway Inhibitor
o Released primary from endothelial cells
o Other sources of TFPT - platelets, mononuclear cells, cardiac myocytes, fibroblasts, vascular smooth muscle, megakaryocytic.
o Inhibits FVIIa-TF complex
o Inhibits FXa with protein S as cofactor
o Decreased TFPI - worsens coagulopathy due to protein S deficiency in people.
Fibrinolysis in health and disease
o Circulating plasminogen is incorporated in the clot and converted to plasmin via tPA and urokinase (fibrinolytic activators)
o Plasmin will break down the fibrin meshwork of the clot
o tPA and urokinase - released from endothelial cells upon activation / injury of endothelium.
o Plasminogen effects are decreased by endogenous plasminogen activator inhibitor (PAI-1)
o TNF alpha and IL1B increase PAI-1 > tPA -> balance towards decreased fibrinolysis
T/F Detecting a hyper coagulable state can be done easily even before the formation of thrombi
FALSE
o A hypercoagulable state is not identified until a thrombotic event occurs or the patient develops DIC, limiting the opportunity to intervene with specific therapies.
o In fact, detecting the presence of a thrombus or thromboembolus is one of the only means for a clinician to learn definitively that pathologic coagulation is occurring.
How can PT/PTT/PLT help in identification of abnormal coagulation?
o Traditional coagulation tests, such as PLT count, activated partial thromboplastin time (aPTT), and prothrombin time (PT), are most accurate for hypocoagulability and do not reliably identify a predisposition towards hypercoagulability.
o Prolongations of aPTT/PT and decreased platelet count may appear in patients with hypercoagulability, although this usually is due to consumption of platelets and coagulation factors after unregulated thrombin generation.
o In practice, a drop in circulating platelet count accompanied by a prolongation of at least 20% in baseline aPTT in an at-risk patient should raise concern of consumptive coagulopathy and prompt further investigation.
How can we document hypercoagulable states?
o Identifying a rise in procoagulant elements (MPs, fV, or VIII activities, or fibrinogen)
o Identifying a decrease in endogenous anticoagulants (AT, protein S and C, or TFPI)
o Identifying a decrease in fibrinolysis (decreased tPA; increased α2-antiplasmin, PAI-1, TAFI)
o Markers of ongoing thrombin generation (thrombin-AT complex [TAT], prothrombin activation fragment [F1+2], or fibrinopeptides A and B) or lysis of fibrin clots (fibrin [-ogen] degradation product [FDP] or D-dimer) may be used.
What are the main viscoelastic coagulation devices and what do they measure?
o TEG and ROTEM
o They evaluates the time to initial fibrin cross-linking, rate of clot formation, and the viscoelastic characteristics of the clot formed.
o Hypercoagulable samples clot more quickly, with a faster rate of clot formation
What is calibrated automated thrombography (CAP)?
o A coagulation test that focuses on the thrombin generation potential (endogenous thrombin potential, ETP) in a sample.
o Hypercoagulable samples exhibit a greater ETP for CAT.
Is there anything we could measure on PLT to see signs of hypercoagulability?
o Platelet contributions to a hypercoagulable state may be inferred by assessing markers of platelet activation (P-selectin expression, platelet-neutrophil aggregates) or documentation of hyperfunctional platelets in response to standard stimuli.
o Detection of specific proteins on platelets or other cir- culating cells requires advanced techniques such as flow cytometry.
o Flow cytometric techniques also can be used to document the pres- ence of procoagulant MPs
Name markers of hyper coagulable state (10)
Name 10 markers of PLT activation
Overview of interaction inflammation / coagulation in inflammatory conditions (sepsis/SIRS)
o Many of the processes by which inflammation affects coagulation are interrelated
o Glycocalyx shedding and EC activation leads to compromised production of local regulators (e.g., NO) and increased expression of procoagulant molecules (e.g., UL-vWF or TF) and adhesion molecules (e.g., P-selectin), with derangement of anticoagulant defenses.
o TM may be damaged by multiple mechanisms (leading to decreased activation of protein C), and AT is less effective because of decreased concentrations and impaired interactions with an endothelium that has been denuded of GAGs.
o TFPI similarly may have impaired EC localization.
o In addition, an exuberant release of PAI-1 resulting from inflammatory cytokine release can slow fibrinolysis and further impede coagulation defenses
Explain the coagulation phases that a septic/SIRS patient will go through
o Patients with sepsis develop an initial hypercoagulable phase, followed by a much longer hypocoagulable phase resulting from consumption.
o The majority of patients described in the veterinary literature display a hypocoagulable phenotype with evidence of prior clot formation.
o In dogs with septic peritonitis, the presence of coagulopathy (defined by prolongations of PT or aPTT, or a platelet count of 100,000/μl or less) is associated independently with increased odds of death.
o Although less is known about coagulopathy in cats, inflammatory conditions (pancreatitis and sepsis) are recognized as two of the top three identified causes of DIC in cats.
o Dogs with sepsis have significantly prolonged aPTT and/or PT, along with higher FDP and D-dimer concentrations than control dogs.
o Septic dogs also have lower protein C and AT activities, further supporting a consumptive coagulopathy. Septic dogs with continually decreasing levels of protein C and AT proteins had a worse outcome.
o TAFI is increased in dogs with bacterial sepsis and other inflammatory conditions (e.g., neoplasia), resulting in downregulation of fibrinolysis.
T/F Dogs with glomerular disease and significant proteinuria with or without nephrotic syndrome (NS) are at a heightened risk of thrombotic complications
TRUE
Why is protein losing nephropathy associated with hypercoagulable states?
o In people, the thrombophilia associated with PLN appears to be multifactorial.
o Platelets are hyperaggregable and exhibit increased markers of activation (P-selectin).
o Soluble factors show increases in fVIII activity and fibrinogen concentration, whereas vWF levels and fV are elevated variably.
o The loss of endogenous anticoagulant potential centers on low AT activity, which occurs in people and dogs.
o Despite this consistent finding, AT activity fails to uniformly predict thrombotic risk across studies in people.
o Protein C levels are variable in patients with PLN, and several studies have documented elevated levels of TFPI, suggesting that this anticoagulant is not likely a significant component of the thrombophilia.
o In people, levels of TAFI can be increased, along with PAI-1, suggesting a decreased fibrinolytic state.
o People have a propensity toward development of renal vein thrombosis, and increased markers of endothelial activation have been documented.
o These suggest some involvement of a local mechanism (e.g., endothelial activation or abnormal renal blood flow) contributing to the overall thrombophilia.
What coagulation abnormalities have been identified in dogs with PLN?
o TEG tracings more hyper coagulable
o Increased fibrinogen, alpha2-antiplasmin and protein C activities
o Decreased AT activity
Thrombi have been identified in up to __% to __% of nonsurvivors and DIC in __% of dogs suffering from IMHA
46% to 80%
45%
What coagulation abnormalities have been detected in IMHA patients?
o Coagulation abnormalities consistent with a hypercoagulable state
o Low AT activity, elevated FDPs and D-dimer, and markedly elevated fibrinogen concentration
o TEG studies have documented hypercoagulability, primarily on the basis of an increased clot strength (maximal amplitude or MA). Fibrinogen, platelet count and function, and hematocrit are key contributors to the MA.
o Circulating TF is also a likely contributor -> upregulation of TF gene expression in whole blood, although the source of the TF has not been determined. Increased TF could come from numerous sources or from stimulation of EC TF expression by cell- free heme.
o Free heme can also decrease the bioavailability of NO and upregulate EC adhesion molecules (e.g., E-selectin).
o Hemolyzed erythrocytes augment thrombin generation in vitro, an effect attributed to erythrocyte-derived MPs or procoagulant erythrocyte membrane
T/F Hyperadrenocorticism in people is associated with a significantly increased risk of thrombotic complications, with rates comparable to those following major orthopedic surgery (rates of venous TE up to 5%)
TRUE
What changes in coagulation have been observed in people with hyperadrenocroticism?
o Elevated activities of fVIII and vWF, heightened levels of PAI- 1 and elevated activities of factors IX, XI, and XII.
o In contrast to veterinary patients, many people with HAC suffer from comorbidities (e.g., obesity, diabetes mellitus, and hypertriglyceridemia) that are also prothrombotic conditions.
o Dogs with HAC are represented in most case series describing thrombotic conditions (aortic thrombosis, PTE, splenic or portal vein thrombosis). Despite these observations, a consistent cause or definable procoagulant state has not been identified.
T/F Thrombosis secondary to cardiac disease is common in dogs
FALSE
o Thrombosis secondary to cardiac disease is reported infrequently in dogs but has been associated with dilated cardiomyopathies and atrial fibrillation.
Ho will affect LA enlargement to coagulation?
o Left atrial (LA) and LA appendage enlargement is associated with numerous structural changes, culminating in a procoagulant phenotype, such as increased TF and vWF on areas of denuded or damaged endothelium.
o Growth hormones (e.g., VEGF), which are increased in people with AF, may promote the upregulation of TF.
o Through atrial enlargement, shear stress is decreased (stasis), reducing the release of NO.
Cardiomyopathic cats and coagulation
o A systemic hypercoagulable state occurs in 50% of cardiomyopathic cats with spontaneous echocardiographic contrast (or “smoke”) with or without a LA thrombus, and in 56% of cats with ATE and LA enlargement.
o vWF : Ag (vonWillebrand factor antigen) concentrations were elevated in only the cats with ATE, and the presence of hypercoagulability was not related to LA size or the presence of congestive heart failure.
o These results are echoed by an earlier study that revealed changes consistent with a hypercoagulable state in 45% of cats with hypertrophic cardiomyopathy.
o Platelets from cats with cardiomyopathy required significantly lower doses of ADP to result in irreversible aggregation compared with control cats.
DIC has been described in __% of dogs with malignancies
9.6%
Higher rates of DIC occur in which type of neoplasias?
Hemangiosarcoma
Mammary carcinoma
Adenocarcinoma of the lung
TF and patients with neoplasia
o TF has been identified on malignant cells and in tumor vasculature, and tumor cells have the ability to shed TF-bearing MPs.
o TF supports thrombophilia and also plays a key role in regulation of integrin function responsible for tumor angiogenesis.
o In mice, TF blockade results in decreased angiogenesisand tumor growth, through modulation of VEGF.
o TF expression on histopathology samples is an independent predictor of poor overall or relapse-free survival for many tumor types in people.
o TF expression has been evaluated in canine cell lines of mammary tumors, pancreatic carcinoma, pulmonary adenocarcinoma, prostatic carcinoma, and sarcomas (osteosarcoma and fibrosarcoma). TF was highly expressed in all but osteosarcoma;
o Tumors of epithelial origin (mammary carcinoma and pulmonary adenocarcinoma) expressed the highest levels. These tumors also shed TF-bearing microparticles into tissue culture supernatants.
Patients with distant metastasis commonly have a higher _______ and ________ compared with locally invasive or noninvasive disease.
Fibrinogen and D-dimer
T/F Platelet and fibrinogen survival in dogs with metastatic disease are decreased, supporting ongoing consumption
TRUE
T/F A state of intravascular coagulation resembling DIC has been recognized in people suffering traumatic brain injury, with significant impacts on outcome
TRUE
The overall mortality for TBI patients with coagulopathy was ___% compared with ____ in patients without coagulopathy.
50.4%
17.3%
What is the brain rich in, that could initiate coagulation?
Tissue factor
TBI and coagulation
o TBI patients have elevated monocyte TF expression for the first 24 hours, which then quickly returns to normal.
o Enhanced thrombin generation has been documented as blood passes the vasculature of the brain. In a study of people with severe isolated TBI, patients had prolonged aPTT and PT; elevated D-dimer, TAT, and F1+2; and low AT, platelets, and fibrinogen upon presentation.
o Procoagulant MPs after TBI are increased significantly in CSF and blood. These MPs were primarily of EC and platelet origin, adding evidence to the likely contribution of cerebrovascular endothelial activation or injury.
o Although local procoagulant factors initiate coagulation, inflammatory cytokines and procoagulant MPs provide a means for dissemination of the condition, leading to a systemic response.
o Studies have suggested a state of platelet hypofunction in brain injured patients. This is opposed to non–brain-injured trauma patients who generally have increased platelet reactivity.
o The cause of the platelet dysfunction in TBI patients has not been identified.
o Eight experimental cats with TBI-induced coagulopathy secondary to bullet-inflicted brain injury showed a decreased platelet count and decreased platelet clumping, possibly suggesting a decreased reactivity of the cats’ platelets. A decreasing fibrinogen was also present throughout the experiment.
What is the main therapy of managing a hypercoagulable state?
Treat the underlying condition!
What recombinant anticoagulant therapies have been investigated to treat hyper coagulable states?
o Antithrombin -> mixed results in people
o Recombinant activated protein C (rAPC) -> benefits experimentally but not in people, removed from market.
o Recombinant TFPI (rTFPI) -> benefits experimentally, not in clinical trials.
o Recombinant soluble TM (rTM) -> maybe benefit on survival, unknown.
Which type of exogenous antithrombotic therapies can we administer to our patients?
o Drugs that inhibit PLT function -> aspirin or clopidogrel
o Drugs that inhibit thrombin formation -> UFH, LMWH
T/F Adjusted-dose heparin therapy (targeting an anti-Xa activity) may improve survival from IMHA by limiting thrombotic complications
TRUE
What is a bleeding disorder?
o Bleeding disorders are conditions that result in inappropriate hemostasis, causing or predisposing to bleeding.
o Some coagulopathies result in spontaneous bleeding, but many are subclinical and hemorrhage occurs only after an invasive procedure.
Primary, secondary hemostasis and fibrinolysis
o Primary hemostasis, involving the interaction between platelets and endothelium resulting in the formation of a platelet plug.
o Secondary hemostasis, a system of proteolytic reactions involving coagulation factors and resulting in the generation of fibrin polymers, which stabilize the platelet plug to form a mature thrombus.
o Fibrinolysis is the dissolution of the fibrin clot to restore vascular patency.
What happens with the platelets when there is a disruption in the endothelium?
o Primary hemostasis immediately follows vascular damage. Platelets adhere to subendothelial collagen via the platelet GPVI receptor, or to collagen-bound von Willebrand factor (vWF) via the GPIb receptor.
o Adherence triggers a cascade of cytosolic signaling that stimulates platelet arachidonic acid metabolism and the release of granular contents (activation).
o Thrombin, generated by secondary hemostasis, is also a powerful platelet agonist.
o Activated platelets release secondary agonists, TxA2, ADP, and serotonin, which recruit and activate additional platelets, thus amplifying and sustaining the initial response.
o The final common pathway for all agonists is the activation of the platelet integrin αIIbβ3 receptor (formerly known as glycoprotein IIbIIIa receptor). Agonist binding induces a conformational change in the receptor, exposing binding domains for fibrinogen.
o Binding results in interplatelet cohesion and aggregation. Aggregated platelets constitute the primary hemostatic plug and provide a stimulus and framework for secondary hemostasis.
T/F Although deficiencies of fXII cause marked coagulation test prolongation, they do not result in a bleeding tendency
TRUE
How easy it is to do hemostatic testing?
o In vitro tests do not accurately reflect in vivo hemostasis.
o Hemostatic testing makes high demands on sampling procedure; improper technique leads to artifactual results.
o Tests should always be performed and interpreted carefully, along with the clinical findings, and with their limitations in mind.
Normal values for common coagulation tests
What is thrombocytopenia? And pseudothrombocytopenia?
o Platelet counts detect quantitative platelet disorders (thrombocytopenia, low PLT number).
o Pseudothrombocytopenia is a common artifact that occurs when platelets in blood are not adequately counted. This usually results from platelet aggregation during sample collection and is especially common in cats.
o Even in the absence of platelet clumping, pseudothrombocytopenia is frequent with automated PLT counts in cats because of the considerable overlap between erythrocyte and PLT volumes in this species, and in dogs and cats when large platelets are present.
o For these reasons, low PLT counts should always be confirmed by blood smear examination.
What is the buccal mucosal bleeding time (BMBT)?
o The bleeding time is the duration of hemorrhage resulting from the infliction of a small standardized injury involving only microscopic vessels and reflects in vivo primary hemostasis.
o The buccal mucosal bleeding time (BMBT) is the only reliable and reproducible method in small animals.
Technique to perform a BMBT?
o Sedation is generally not required, except in cats and nervous dogs.
o The patient is restrained in lateral recumbency and a strip of gauze is tied around the maxilla to fold up the upper lip, sufficiently tight to cause moderate mucosal engorgement.
o A spring-loaded device is used to make two 1-mm–deep incisions in the mucosa of the upper lip.
o The incisions should be made at a site devoid of visible vessels and inclined so that the blood flows toward the mouth.
o Shed blood is blotted carefully with filter paper, taking extreme care not to disturb the incisions.
o The BMBT is the time from incision to cessation of bleeding.
When is a BMBT indicated?
o It is indicated in patients with a suspected primary hemostatic defect when the PLT count is adequate.
o It is prolonged in dogs with von Willebrand disease (vWD) and nonsteroidal antiinflammatory drug –induced thrombopathia.
o The BMBT also is used for the preoperative screening of patients considered at risk for vWD or other thrombopathias.
Limitations of BMBT?
o It is influenced by hematocrit and blood viscosity and has appreciable inter- and intraoperator variability (up to 2 minutes).
o It is a poor predictor of surgical bleeding.
What does the prothrombin time measures?
o It evaluates the extrinsic and common pathways, specifically factors II, V, VII, X and fibrinogen.
o Because of the short half-life of factor VII, the PT is sensitive to vitamin K deficiency or antagonism.
Vitamin K dependent coagulation factors
o II, VII, IX and X
o Protein C and protein S
What does the aTTP measure?
o The APTT evaluates the intrinsic and common pathways.
o Only factors VII and XIII are not evaluated.
o It is more sensitive to heparin than is the PT.
How good are PT/PTT values at predicting bleeding
o They are in vitro plasma-based tests, represented by the cascade model of coagulation, and do not accurately represent in vivo hemostasis.
o As such, they are not predictive of bleeding.
When are fibrin degradation products generated?
They are generated when fibrinogen, soluble fibrin, or cross-linked fibrin is lysed by plasmin.
What does increased FDPs means?
o Elevated concentrations indicate increased fibrinolysis and/or fibrinogenolysis.
o Because clearance is by hepatic metabolism and the mononuclear phagocytic system, disorders of these systems also result in elevated FSP concentrations.
o FDPs can inhibit coagulation and induce platelet dysfunction, contributing to a bleeding tendency.
o They are commonly detected with DIC but are not specific for the condition; elevated concentrations are also described in dogs with thromboembolism (TE), neoplasia, IMHA, hepatic dysfunction, sepsis/SIRS, heat stroke, trauma, heart failure, and GDV.
When are D dimers produced?
When fibrin is cross linked by FXIIIa
What is the difference between d-dimers and other FDPs?
o In contrast to other FSPs, which indicate only the activation of plasmin, D-dimers indicate the activation of thrombin and plasmin and are specific for active coagulation and fibrinolysis.
o The half-life of D-dimers is short (approx. 5 hours); therefore elevated concentrations indicate recent or ongoing fibrinolysis.
How should we interpret d-dimers results?
o D-dimers are a sensitive indicator of thrombotic conditions, such as DIC and thromboembo- lism (TE), and are more sensitive than are FSPs.
o They have good negative predictive value, but the absence of elevated D-dimers does not preclude a diagnosis of DIC.
o Conversely, elevated D-dimer concentrations are not specific; elevated concentrations are demonstrated in dogs with DIC, TE, neoplasia, hepatic disease, renal failure, cardiac failure, internal hemorrhage, and after surgical procedures.
How do we normally measure fibrinogen concentrations and how should we interpret the results?
o Fibrinogen concentration is usually determined via the Clauss method, a functional assay based on the time for fibrin clot formation after the addition of excess thrombin.
o Decreased concentrations (hypofibrinogenemia) can be inherited or acquired.
o Acquired disorders are described with hemodilution, massive transfusion, hepatic dysfunction, DIC, and sepsis and after thrombolytic therapy.
o Hypofibrinogenemia generally does not result in prolongation of standard coagulation tests (PT, aPTT) until fibrinogen is markedly decreased (less than 50 to 100 mg/dl).
Thrombin time
o The thrombin time (TT) tests functional fibrinogen, via measure of the time taken for a standardized thrombin solution to convert fibrinogen to fibrin.
o The TT is prolonged with hypofibrinogenemia, dysfibrinogenemia, or in the presence of factors that inhibit fibrin polymerization (e.g., heparin, FDPs).
What do thromboelastography (TEG) and thromboelastometry (ROTEM) measure?
o The viscoelastic properties of the blood clot are evaluated, from initiation of coagulation, through amplification and propagation, to fibrinolysis.
o Information is generated regarding the strength and stability of the clot and the dynamics of its formation and breakdown.
o Compared with routine hemostatic tests, these methodologies provide global assessment of hemostasis as determined by the interplay of plasma and cellular components, more closely reflecting in vivo hemostasis.
How does a TEG assay works?
o The thromboelastograph consists of a plastic cup and a pin suspended by a torsion wire.
o A sample of citrated blood is placed in the cup with calcium chloride (at 37° C), and the cup is elevated so that the pin hangs in the sample.
o The cup is then oscillated around the vertical axis.
o When fibrin strands form between the pin and the cup, the torque (twisting force) generated is transmitted to a transducer, which converts the signal data for computer display of the TEG tracing.
o Testing is routinely performed 30 minutes after sampling. Reliable and reproducible results also can be obtained at 120 minutes, but results are statistically different from 30 minutes.
What parameters will we obtain with a TEG and what do they mean?
o The reaction time (R) represents the enzymatic portion of coagulation (secondary hemostasis).
o The clotting time (K) represents clot kinetics, largely determined by clotting factors, fibrinogen, and platelets.
o The angle (α) depends largely on fibrinogen, as well as platelets and factors.
o The maximum amplitude (MA) represents the ultimate strength of the fibrin clot, dependent primarily on platelet aggregation (platelet number and function) and, to a lesser extent, fibrinogen.
o MA is used to derive the clot shear elastic modulus G, where G=5000×MA/(100−MA) and is a measure of the overall coagulant status.
o Fibrinolysis is measured by the extent of clot lysis at 30 and 60 minutes after MA (LY30 and LY60, respectively).
Which type of samples can be used for TEG?
o Naive citrated samples
o Samples activated with recombinant human TF
In a prospective study, TEG correctly identified bleeding, with a positive predictive value (PPV) of __% and a negative predictive value (NPV) of __%, based on G alone.
89%
98%
T/F In human patients, TEG has shown clinical utility in predicting bleeding and guiding transfusion therapy.
TRUE
What information does ROTEM provide?
Similar to TEG, ROTEM provides information on initial fibrin formation (clotting time, CT), kinetics of fibrin formation (clotting formation time, CFT; angle, α), maximum fibrin clot strength (maximum clot firmness, MCF), and clot lysis at 30 and 60 minutes (CL30 and CL60)
What are limitations of TEG and ROTEM?
o Limitations of TEG and ROTEM are the inability to detect vWD and insensitivity to antiplatelet drugs.
o The methodologies are also affected by blood viscosity; polycythemia results in hypocoagulable tracings and anemia produces hypercoagulable tracings.
How do we classify hemostatic disorders?
o They are classified as disorders of primary or secondary hemostasis, or both, based on the pathophysiology of the hemostatic defect.
o Disorders of primary hemostasis result from decreased circulating platelet numbers (thrombocytopenia), from platelet dysfunction (thrombopathia) or, rarely, from a vascular anomaly (vasculopathy).
o Disorders of secondary hemostasis result from low concentration or activity of coagulation factors.
o Inherited disorders are almost invariably a single defect in the hemostatic mechanism, whereas acquired disorders, which are more common, frequently affect more than one aspect of hemostasis.
o Disorders of fibrinolysis (hyperfibrinolysis) can also cause, or contribute to, clinical bleeding. Hyperfibrinolysis is demonstrated in DIC and in massive trauma and is suspected to be the primary mechanism of delayed postoperative bleeding in Greyhound dogs.
Examples of primary hemostasis disorders
Disorders of secondary hemostasis
T/F New-onset thrombocytopenia is an independent predictor of ICU mortality, and the severity of thrombocytopenia and the extent of decrease in platelet count are inversely related to survival.
TRUE
What is dilutional coagulopathy?
o Refers to the syndrome resulting from blood loss, consumption of coagulation factors and platelets, and intravascular volume replacement.
o During hypovolemic shock, reduced intravascular hydrostatic pressure results in shifts of coagulation factor-deficient interstitial fluids into the plasma.
o This is compounded by aggressive resuscitation with IV fluids and/or massive red blood cell transfusion and further exacerbated by synthetic colloids, particularly hydroxyethyl starches (hetastarch).
Studies in humans have found that __% to __% hemodilution is required to produce a coagulopathy and that coagulopathy increases with increasing volumes of intravenous fluid administration
40% to 60%
Mechanism of dilutional coagulopathy - 1
o The mechanisms of dilutional coagulopathy are multiple.
o Fibrinogen is the first factor to become critically reduced. Fibrinogen is required in substantially higher concentrations than other factors, but the limited increase in synthesis cannot compensate for increased breakdown.
o The resultant hypofibrinogenemia decreases thrombin formation and fibrin polymerization, decreasing the speed, strength, and stability of clot formation.
o This effect is seen with even moderate blood loss and hemodilution. The magnitude of effect is determined by the severity of the hypofibrinogenemia and the type of resuscitative fluid used.
o At fibrinogen levels below 50 mg/dl no clot is formed; clot formation occurs almost linearly up to 300 mg/dl.
o Hetastarch demonstrates the most pronounced hemodilution effects because it also affects vWF and fVIII.
Mechanism of dilutional coagulopathy - 2
o Thrombocytopenia and the dilution of coagulation factors affect coagulation at a later point in resuscitation than does hypofibrinogenemia.
o After replacement of one blood volume of platelet-deficient fluid, only 35% to 40% of platelets remain in the circulation.
o In the patient with a normal platelet count before resuscitation, this dilution may not be clinically significant.
o Prolongation of PT and aPTT occurs in human patients after replacement of two blood volumes. Prolongations were demonstrated in 70% of dogs after massive transfusion, but a correlation to transfused volumes was not made.
o Fibrinolysis is affected by hemodilution. Progressive dilution of α2-antiplasmin and fXIII reduces fibrin cross-linking and prolongs the half-life of plasmin.
o Plasminogen activator inhibitor is also decreased, resulting in prolonged tPA activity. The net result is enhanced fibrinolysis.
Hypothermia effects of coagulation
o Hypothermia, as results from hypoperfusion, evaporation from exposed body cavities during surgery, or the infusion of cold resuscitation fluids, leads to a reversible hypocoagulability.
o Platelets are extremely temperature sensitive.
o Evidence suggests that the bleeding tendency observed in humans at mildly reduced temperatures (33° to 37° C) results primarily from decreased vWF-mediated platelet adhesion.
o At temperatures below 33° C, reduced platelet function and enzyme activity occur, with TF-fVIIa complex activity decreasing linearly.
o Because conventional coagulation tests are performed at 37° C hypothermia-induced coagulopathy may be difficult to detect.
Acidemia effects on coagulation
o Acidemia, as occurs with hypoperfusion or massive transfusion of CPDA-stored red cells, results in increased fibrinogen degradation and impaired coagulation protein activity.
o fXa-Va complex activity is decreased by 50% at pH 7.2, and 70% at pH 7.0.
o At a pH of 7.0, factor VIIa activity is also decreased by more than 90%.
o The coagulopathy is not reversed with correction via buffer administration.
What are the main 3 questions we should try to answer when we have a bleeding patient?
1) Does the patient have a bleeding disorder or is bleeding the result of local factors?
2) If the patient does have a bleeding disorder, what is the nature of the hemostatic defect: primary hemostasis, secondary hemostasis, fibrinolysis, or a combination of these?
3) Is the defect inherited or acquired? These questions usually can be answered easily based on information gleaned from the history, physical examination, and routine hemostatic testing.
How can the signalment be useful in a bleeding patient?
o Severe inherited disorders are generally apparent within the first 6 months of life.
o Milder forms, such as vWD, may not be diagnosed until surgery, trauma, or concurrent disease intervenes.
o A history of repeated bleeding episodes suggests an inherited disorder.
Which conditions can produce spontaneous bleeding?
o Some inherited disorders (e.g., hemophilia) and many acquired disorders (e.g., thrombocytopenia, vitamin K deficiency) produce spontaneous bleeding.
o Other conditions (e.g., vWD, factor VII deficiency, fibrinolytic disorders) more commonly require some form of trauma to make the impairment apparent.
How is the bleeding presented in a patient with primary hemostasis defects? And with secondary hemostasis defects?
o Defects of primary hemostasis are characterized by ecchymosis and/or spontaneous bleeding from mucosal surfaces (e.g., epistaxis, gingival bleeding, hyphema, hematuria, melena). Petechiae are typical of thrombocytopenia rather than thrombopathia.
o Defects of secondary hemostasis are usually characterized by single or multiple hematomas and bleeding into SQ tissue, body cavities, muscles, or joints.
o Some acquired disorders, such as DIC, defy this classification because multiple hemostatic defects are present.
o vWD usually has the characteristics of a primary hemostatic defect but, in its most severe form, may mimic a secondary hemostatic disorder.
What diagnostics should we do in a bleeding patient?
o An initial diagnostic panel should include, at minimum, platelet enumeration/estimation, PT, and aPTT.
o D-dimer and fibrinogen concentrations are also recommended.
o This testing is generally sufficient to confirm a hemostatic defect and to characterize the defect as a disorder of primary hemostasis, secondary hemostasis, or both.
o With characterization of the hemostatic defects, a concise list of differential diagnoses can be constructed and further diagnostic workup efficiently pursued.
Basic principles of management of a bleeding patient?
- Recognize and treat shock and any other life-threatening conditions.
- Control local bleeding, if possible.
- Restore normal hemostasis via blood product transfusion, medications, reversal of hypothermia, and/or control of other precipitating or contributing factors.
- Monitor for stability of coagulation parameters; correct as needed.
- Monitor for new or ongoing sources of blood loss.
- Monitor for complications associated with new or ongoing blood loss (e.g., intrapulmonary hemorrhage).
T/F Because shock and fluid resuscitation can exacerbate coagulopathy, all attempts should be made to limit the duration of shock and to aggressively correct the coagulopathy while reducing hypoperfusion, hemodilution, and hypothermia
TRUE
When is FFP indicated?
o It contains hemostatic factors equivalent to the plasma from which it was obtained and is indicated for the treatment and prevention of bleeding associated with acquired and inherited disorders of secondary hemostasis.
o An exception is heparin-induced bleeding, because the hemorrhagic diathesis is caused by factor inhibition, not deficiency; moreover, antithrombin in FFP may enhance heparin effects.
What does cryoprecipate contains?
o Cryoprecipitate is prepared from FFP and contains fVIII, vWF, fibrinogen, and fibronectin in 10% of the original plasma volume.
o CP is indicated for the management of patients with vWD, factor VIII deficiency, hypofibrinogenemia, and dysfibrinogenemia.
o Strong evidence in human patients indicates a beneficial role of CP in the management of dilutional coagulopathy and trauma-induced coagulopathy
If we do not have access of platelet concentrates, what can we administer to our patient to provide platelets?
Fresh whole blood
When should we consider a platelet transfusion?
o Platelet transfusion in veterinary medicine is usually therapeutic, indicated for the management of uncontrolled or life-threatening bleeding resulting from severe thrombocytopenia or thrombopathia.
o Even with immune-mediated thrombocytopenia (ITP), in which transfused platelets are rapidly destroyed, a negligible increase in platelet count may provide adequate, life-saving hemostasis.
o Prophylactic platelet transfusions should be considered in dogs with severe thrombocytopenia or thrombopathia before surgery.
Name prohemostatic agents?
DDAVP
TXA
EACA
DDAVP as a prohemostatic agent
o Desmopressin is a synthetic vasopressin analog that induces, via V2 receptors, the release of subendothelial vWF stores.
o DDAVP is used for the management of thrombopathia of various causes in humans. In dogs, DDAVP is used as adjunctive treatment of bleeding associated with canine type 1 vWD, as well as for presurgical prophylaxis (administered 30 minutes before surgery).
o Injectable or intranasal DDAVP is administered at 1 to 4 mcg/kg SC or IV. Onset of action is delayed approximately 30 minutes, and duration of effect is usually 2 hours. The effects of repeated doses are diminished because vWF stores are depleted.
o The efficacy of DDAVP is variable and its effects short- lived; the patient should be closely monitored and blood products made available.
Epsilon aminocaproic acid and tranaxemic acid as prohemostatic agents
o Epsilon- aminocaproic acid (EACA) and tranexamic acid (TEA) are lysine analogs; they block the binding and activation of plasminogen and also exert antiinflammatory effects through interleukin inhibition.
o TXA is approximately 10 times more potent than EACA.
o TXA and EACA appear to have comparable efficacy with minimal risk of adverse events
o EACA neutralizes experimentally induced hyperfibrinolysis in dogs and has a wide therapeutic index.
o EACA has been shown to reduce postoperative bleeding in Greyhound dogs when administered preemptively at 15 to 40 mg/kg q8h.
How can we confirm thrombocytopenia?
Thrombocytopenia is confirmed by a low platelet count, verified by blood smear examination.
Can we predict if our patient will bleed or not based on a PLT count?
o No
o The bleeding threshold is not predictable from the count alone and depends on factors such as platelet function, secondary hemostasis, and the presence of precipitating trauma.
o Spontaneous bleeding generally does not occur until platelet counts fall below 30,000 to 50,000/μl, unless a concomitant bleeding disorder exists.
o In the patient with spontaneous bleeding, a platelet count of more than 50,000/μl should prompt investigation for another contributing hemostatic defect.
o Platelet counts as low as 5000/μl can occur without bleeding.
Patients with ITP tend to bleed less or more than other patients with equivalent PLT counts?
o Less
o Patients with ITP tend to bleed less than patients with equivalent counts from other causes because of the presence of young, hyperfunctional platelets.
T/F Thrombocytopenia associated with splenic torsion, although it can be profound, is not associated with bleeding.
TRUE
Nonpathologic thrombocytopenia is reported in ______ _______ _______ _______ and ___________ dogs and should not be overinterpreted
Cavalier King Charles Spaniels
Greyhound
Thrombopathia
o Vascular disorders are an uncommon cause of bleeding.
o In the patient with a primary hemostatic disorder and adequate platelet numbers, a platelet function defect is likely.
o A prolonged BMBT in a patient with adequate platelet count confirms thrombopathia.
o The drug history should be carefully appraised because numerous drugs can cause or contribute to thrombopathia.
o Diseases known to affect platelet function should be excluded. If no obvious cause of acquired thrombopathia can be found, a hereditary disorder is suspected.
What type of inherited coagulopathy has been described in Devon Rex cats?
Deficiency of vitamin K dependent factors (II, VII, IX and X)
T/F Most inherited coagulopathies cause bleeding within the first year of life
TRUE
What test will be increased with FVII deficiency? And with FVIII and FIX (hemophilia)?
PT
aPTT
With which factors coagulopathies will both PT and aPTT be increased?
FI
FII
FX
And with vitamin K factors deficiency
When is cryoprecipitate indicate? And cryosupernatant?
o Cryoprecipitate is ideal for deficiencies of factors VIII and fibrinogen.
o Cryosupernatant is indicated for deficiencies of factors II, VII, IX, X, and XI.
o Where these products are not available, FFP is an acceptable option.
FXII deficiency
o Factor XII deficiency is an asymptomatic condition of dogs and cats. It is the most common factor deficiency in cats, with a reported prevalence of 2.1%.
o Because FXII is involved in contact factor activation but is not essential for in vivo hemostasis, deficiency results in significant prolongation of the aPTT but no hemorrhagic tendency.
o FXII deficiency is usually diagnosed incidentally and must be distinguished from pathologic causes of aPTT prolongation.
An absolute or relative vitamin K deficiency occurs in which conditions?
o Dietary insufficiency is rare but has been reported in neonates or with prolonged TPN administration.
o Broad-spectrum oral antimicrobial drugs can inhibit vitamin K synthesis.
o Decreased absorption can result from severe gastrointestinal disease, hepatopathy, pancreatic insufficiency, or biliary obstruction.
o Vitamin K antagonism occurs with warfarin therapy or anticoagulant rodenticide toxicity. These compounds inhibit vitamin K epoxide reductase, leading to a relative vitamin K deficiency.
Vitamin K pathway
Half-life of FVII
4-6h
With vit K deficiencies, which test gets increased first, PT or aPTT?
o PT prolongation occurs first, reflecting the short half-life of fVII (4 to 6 hours).
o Prolongation of the aPTT follows when other factors are depleted (approximately 2 days).
Why does hepatic disease can manifest coagulopathies?
The liver plays a pivotal role in hemostasis by synthesizing clotting factors, coagulation inhibitors (antithrombin, protein C), and fibrinolytic proteins, as well as by clearing activated factors, enzyme- inhibitor complexes, and FDPs.
Because of the large reserve capacity of the liver, decreased factor synthesis occurs only with significantly decreased functional hepatic mass (more than __%), with F__ showing earliest reduction
70%
fVII
Why can thrombocytopenia occur with liver disease?
Primarily because of impaired hepatic synthesis of thrombopoietin
Is hypofribinogenemia common with hepatic disease?
No
How can we differentiate DIC vs hepatic disease?
o DIC rarely occurs secondary to hepatic disease.
o They have similar hemostatic defects and pattern of laboratory anomalies, therefore distinguishing these conditions can be challenging.
o Fibrinogen levels tend to be lower, and D-dimer concentrations higher, in DIC compared with hepatic disease
T/F Hyperfibrinolysis should be considered in the patient with unrelenting hemorrhage after correction of other measurable parameters.
TRUE
ACoTS vs RAC
o Approximately 25% of severely injured patients are presented with a clinically significant coagulopathy that develops minutes after the initial traumatic insult.
o This coagulopathy, termed the acute coagulopathy of trauma and shock (ACoTS), is associated with a fourfold increase in mortality.
o Resuscitation-associated coagulopathy (RAC) develops later in the posttraumatic period secondary to hypothermia, worsening acidosis, and hemodilution resulting from the administration of intravenous fluids and/or blood transfusion.
Why does ACoTS occur?
o ACoTS is caused by tissue trauma, shock, sympathoadrenal activation, and inflammation.
o Shock with tissue hypoperfusion appears to be the major driver of ACoTS, with the extent of hypoperfusion directly correlated with the degree of coagulopathy.
o Higher injury severity increases the incidence and severity of coagulopathy in hypoperfused patients.
o Fibrinolysis is activated early after injury. Hypoperfusion is believed to result in increased expression of thrombomodulin on the surface of endothelial cells. The resultant increases in activated protein C (APC) inactivate fVIIa and fVa and promote fibrinolysis through the inhibition of plasminogen activator inhibitor-1 (PAI-1).
o In addition, endothelial activation and injury lead to the release of tPA, endogenous heparinization from glycocalyx shedding, and increased vascular permeability. Recent studies show that fibrinogen concentrations are decreased in injured patients on admission, even preceding significant fluid resuscitation, and are associated with poor outcomes.
Why does resuscitation-associated coagulopathy happens?
o RAC occurs in the period after injury, as a result of persistent hypothermia, acidemia, and hemodilution.
o These effects result in multiple hemostatic derangements that include hypofibrinogenemia, platelet dysfunction, thrombocytopenia, and decreased enzymatic activity.
Diagnostics to detect trauma-induced coagulopathy?
o Traditional plasma-based coagulation tests, such as the PT and aPTT, do not accurately reflect the in vivo coagulopathy.
o The PT appears to be more sensitive than the aPTT in identifying enzymatic disorders in these patients, but these tests do not detect hyperfibrinolysis or platelet dysfunction and are not prolonged until fibrinogen falls to exceedingly low levels.
o Viscoelastic testing (TEG and ROTEM) have proved superior in predicting coagulopathy and in guiding plasma and platelet transfusions.
Permissive hypotension and trauma-induced coagulopathy
o Newer protocols, termed “damage control resuscitation,” focus on the prevention of coagulopathy through permissive hypotension, limiting fluids, and delivering higher ratios of plasma and platelets.
o The goal of permissive hypotension is to minimize dilutional coagulopathy secondary to fluid administration by maintaining a lower systemic blood pressure.
o In humans with penetrating injuries, maintaining a target MAP of 50 mm Hg, compared with 65 mm Hg, was associated with a decreased incidence of coagulopathy, decreased blood product use, and improved survival.
o Intravascular volume support is achieved preferentially via red cell and FFP transfusion, with plasma administered as early as possible. Several studies have shown the clinical benefit of aggressive hemostatic resuscitation using the empiric transfusion ratio of FFP:RBC over 1:1.
Studies have shown that fibrinogen concentrations less than________ to be highly predictive for hemorrhage.
200mg/dl
What is DIC
o DIC is characterized by the systemic activation of coagulation, leading to widespread microvascular thrombosis, which compromises organ perfusion and can contribute to organ failure.
o The ongoing activation of coagulation may exhaust platelet and coagulation factors, resulting in a hypocoagulable state and bleeding.
What causes DIC?
DIC invariably occurs as a complication of an underlying disorder; sepsis and the systemic inflammatory response syndrome (SIRS) are the most common causes in humans and dogs
Clinical signs of DIC
o It can range from asymptomatic (nonovert DIC) to signs of organ failure associated with microvascular thrombosis to fulminant bleeding (overt DIC).
o Bleeding occurs in a minority of patients with DIC; organ dysfunction is more common.
Hemostatic evaluation via TEG showed that the majority of dogs with DIC were ________; only __% were ___________.
Hypercoagulable
22%
Hypocoagulable
Diagnosis of DIC?
o DIC represents a dynamic continuum, and findings depend on where that patient lies on the continuum at that point in time.
o Hemostatic tests are not specific for DIC. Neither a gold standard nor consensus for the diagnosis of DIC exists in animals.
o Hemostatic tests are best evaluated together, and in light of clinical findings. Diagnosis is generally based on the presence of an underlying condition that could trigger DIC, together with three or more of the following anomalies: thrombocytopenia, prolongation of the PT, aPTT or TT, elevated D-dimers, hypofibrinogenemia, reduced antithrombin activity, and/ or evidence of red blood cell fragmentation (schistocytes) on blood smear examination.
o The diagnosis of DIC by routine laboratory testing is restricted to identification of the overt coagulopathic stage of the disease.
o TEG enables identification of the more common hypercoagulable phase. Differentiation between hypercoagulable and hypocoagulable patients has been demonstrated; higher mortality rates occur in hypocoagulable dogs
Delayed postoperative bleeding in Greyhounds
o The prevalence of postoperative bleeding in Greyhound dogs far exceeds that of other breeds. Bleeding rates of 26% have been reported in Greyhounds after routine gonadectomy, compared with 0 to 2% in other dog breeds.
o Bleeding is delayed 36 to 72 hours after surgery.
o In some dogs, bleeding progresses to a generalized bleeding disorder associated with clinical signs of illness, profuse widespread bleeding, mild thrombocytopenia, hemolysis, and increased hepatic and muscle enzyme activities.
o No significant differences have been identified between bleeders and nonbleeders with respect to platelet count, platelet function, PT, aPTT, fibrinogen concentration, D-dimer, factor XIII, and plasminogen.
o Antiplasmin and antithrombin activities have been shown to be significantly lower in dogs that bled compared with those that did not. These findings, together with the delayed onset of the bleeding suggest anomaly of the fibrinolytic system or endothelial dysfunction, rather than a primary or secondary hemostatic disorder.
How can we prevent / treat delayed postoperative bleeding in Greyhounds
o Postoperative bleeding in Greyhound dogs is reduced by the prophylactic use of EACA.
o Dogs that did not receive preemptive EACA after limb amputation were 5.7 times more likely to bleed than dogs that did receive EACA.
o Of 100 elective gonadectomies in retired racing Greyhound dogs, bleeding occurred in 30% of the placebo group compared with only 10% of the EACA group.
o In both studies, EACA was administered at 500- to 1000mg total dose (15 to 40 ml/ kg) every 8 hours for 5 days, beginning immediately or soon after surgery. An increased likelihood of bleeding associated with body weight suggests that higher dose rates may be more effective.
The presence of thrombocytopenia in veterinary medicine has been associated with decreased survival in patients with ____, _____ and _____ ___________.
IMHA
Neoplasia
Feline panleukopenia
In dogs the underlying diseases in thrombocytopenia patients included ITP (__%); neoplasia-associated thrombocytopenia (__%); inflammatory or infectious causes (__%); and other causes or combined causes of thrombocytopenia (__%).
5%
13%
23%
59%
In 41 cats with thrombocytopenia presenting to a veterinary referral hospital, causes included infectious disease (__%), neoplasia (__%), cardiac disease (_%), and only one cat with primary immune-mediated disease; eight of the cats (__%) did not have a definitive diagnosis.
29%
20%
7%
20%
Infectious causes of thrombocytopenia? And other causes?
o Include tickborne infections such as Rocky Mountain spotted fever, anaplasmosis, babe- siosis, and ehrlichiosis.
o Other commonly incriminated infectious causes include leishmaniasis, leptospirosis, heartworm disease, feline immunodeficiency virus, feline leukemia virus, feline infectious peritonitis, and sepsis.
o Systemic inflammatory diseases, including neoplasia and subsequent DIC, and systemic thrombosis can cause thrombocytopenia.
o Immune-mediated mechanisms cause the most severe form of thrombocytopenia. One study has identified platelet-bound antibodies in thrombocytopenic dogs with a multitude of underlying conditions, including infectious and neoplastic causes, pancreatitis, hepatitis, and SIRS. This suggests many conditions can cause immune-mediated destruction of circulating platelets.
Clinical signs of thrombocytopenia?
o Clinically significant bleeding as a result of low platelet numbers can be observed when platelet count drops below 25 to 50 × 10^9/L.
o Clinical signs of thrombocytopenia include petechiae and ecchymoses, frequently seen on oral mucous membranes, ear pinnae, or in the inguinal area.
o Mucosal bleeding is also common, causing epistaxis, hyphema, hematemesis, melena, hematochezia, and hematuria.
o CNS signs resulting from cerebral and spinal cord bleeds may be seen, and prolonged bleeding or excessive bruising may follow venipuncture or trauma.
T/F In critically ill human patients, thrombocytopenia has been associated with decreased survival; moreover, a decrease in platelet count, even if the platelet count remains within normal range, serves as an independent predictor of mortality
TRUE
T/F Even in the absence of an overt inflammatory condition, patients with critical illness can develop a consumptive coagulopathy and thrombocytopenias.
TRUE
How can thrombocytopenia occur?
1) Decreased production
2) Consumption
3) Sequestration
4) Increased destruction.
T/F Decreased production of platelets at the bone marrow can be either due to a real decrease in production, or a direct destruction of megakaryocytes
TRUE
Causes of suppression / destruction of megacaryocytes?
May be caused by immune-mediated disease, drugs or toxins, infectious agents, irradiation, hypoadrenocorticism, neoplasia, and myelophthisis.
How can we classify drug-induced thrombocytopenia?
1) Decreased production via bone marrow suppression and/or
2) Increased platelet destruction by immune-mediated processes.
o Non–immune- mediated thrombocytopenia develops more gradually as megakaryocytes are suppressed and replacement of senescent platelets falters. Immune-mediated thrombocytopenia likely develops more quickly
How can drugs induce immune-mediated destruction of megakaryocytes?
o By several proposed mechanisms.
o The first is hapten-dependent antibody formation, caused by the drug binding covalently to platelet membrane proteins, causing antibody production and drug-specific immune reaction. Examples of drugs that use this mechanism include penicillins and cephalosporins.
o Other drugs can induce production of antibodies that bind to platelet membrane proteins (most commonly glycoprotein [GP] IIbIIIa/integrin αIIbβ3) in the presence of the drug, or optimize interactions between antibodies and platelet antigen. Examples of drugs that use this mechanism include quinine, vancomycin, sulfonamides, rifampin, and fluoroquinolones.
o Other drugs induce production of auto-antibodies that react with platelets even in the absence of the drug, such as sulfonamides.
Mechanism of non-immune mediated drug induced thrombocytopenia
Non–immune-mediated mechanisms include bone marrow suppression or toxicity; examples include phenobarbital, chloramphenicol, penicillins, cephalosporins, chemotherapeutics, methimazole, azathioprine, and albendazole.
Treatment choices for patients with bleeding resulting from thrombocytopathies
Platelet production
Platelet structure
o Alpha granules - adhesion molecules (P-selectin, vWF), coagulation factors (FV, FVII)
o Dense granules - ADP, histamine, serotonine, Ca2+, Mg2+ epinephrine
o Several protein membranes can be stored in the granules and be expressed with activation
Platelet activation
Major G coupled protein platelet receptors
Major leucine rich receptors (LRR) in PLT and its ligands
Immunoglobulin superfamily PLT receptors
Integrins receptors platelets structure
Integrin receptors and their ligands in PLT
FAT CAT study findings
Findings on this paper
PLT alpha granules
o Alpha granules: 50-80 per PLT (most frequent)
PLT dense granules
o 3-8 per platelet
PLT lysosomes
o 0-3 per platelet
Initiation phase - what will happen once TF binds FVIIa?
o The TF-FVIIa complex then activates additional FVII to FVIIa, allowing for even more TF-FVIIa complex activity, which then activates small amounts of FIX and FX.
o Although it occurs slowly, FV can be activated directly by FXa.
o The FXa generated by TF-FVIIa binds to the few generated molecules of its cofactor FVa to form the prothrombinase complex, which subsequently cleaves prothrombin and generates a small amount of thrombin.
o Any FXa that dissociates from the membrane surface of the TF-bearing cell is rapidly inactivated by either TFPI or AT.
o The FXa generated is consequently effectively restricted to the surface of the TF-bearing cell on which it was generated.
o However, the FIXa generated can dissociate and move to the surface of nearby platelets or other cells. FIXa is not inhibited by TFPI, and much more slowly inhibited by AT than is FXa.
o Because TF is always expressed in the perivascular space, any FVIIa that leaves the vasculature through minor breaks in the endothelial barrier will bind to TF and potentially initiate coagulation.
Amplification phase
o Once a small amount of thrombin has been generated on the surface of a TF-bearing cell (the initiation phase), that thrombin diffuses away from the TF-bearing cell and is available for activation of platelets that have leaked from the vasculature at the site of injury.
o Binding of thrombin to platelet surface receptors causes extreme changes in the surface of the platelet, resulting in shape change, shuffling of membrane phospholipids to create a procoagulant membrane surface, and release of granule contents that provide additional fuel for the fire.
o Platelet granules contain a large number of proteins and other substances that include raw materials for clotting reactions and agonists to induce further platelet activation.
o Calcium may induce clustering of PS (increasing the procoagulant nature of the membrane), and promotes binding of coagulation proteins to the activated membrane surface.
o In addition to activating platelets, the thrombin generated in the initiation phase cleaves FXI to FXIa and activates FV to FVa on the platelet surface.
o Thrombin also cleaves von Willebrand factor off of FVIII (they circulate bound together), releasing it to mediate platelet adhesion and aggregation. The released FVIII is subsequently activated by thrombin to FVIIIa.
Propagation phase
o Once a few platelets are activated in the amplification phase, the release of the granule contents results in recruitment of additional platelets to the site of injury.
o The propagation phase occurs on the surface of these platelets. Expression of ligands on their surface results in cell–cell interactions that lead to aggregation of platelets.
o FIXa that was generated by TF-FVIIa in the initiation phase can bind to FVIIIa (generated in the amplification phase) on the platelet surface. Additional FIXa is generated due to cleavage of FIX by FXIa that was generated during amplification on the platelet surface.
o Once the intrinsic tenase complex forms (FIXa– FVIIIa) on the activated platelet surface, it rapidly begins to generate FXa on the platelet.
o FXa was also generated during the initiation phase on the TF-bearing cell surface. As this FXa is rapidly inhibited if it moves away from the TF-bearing cell surface, it can not easily reach the platelet surface.
o The majority of FXa must therefore be generated directly on the platelet surface through cleavage by the intrinsic tenase complex.
o The FXa generated on platelets then rapidly binds to FVa (generated by thrombin in the amplification phase) and cleaves prothrombin to thrombin.
o This prothrombinase activity results in a burst of thrombin generation leading to cleavage of fibrinopeptide A from fibrinogen. When enough thrombin is generated with enough speed to result in a critical mass of fibrin, these soluble fibrin molecules will spontaneously polymerize into fibrin strands, resulting in an insoluble fibrin matrix.
Blood products, storage guidelines and indications
Do dogs have naturally occurring alloantibodies?
o Yes - naturally occurring anti-DEA 7 antibodies in 50% of DEA-7 negative transfusion naïve dogs.
o Implicated in causing delayed TR
o Not in North America
Blood transfusion guidelines (product, composition, dose, rate - table)
Abnormalities associated with massive transfusion - table
Pre - transfusion serologic tests
Gold standard test for typing?
Major crossmatch
Minor crossmatch
Canine blood types
DEA prevalence
o No longer DEA 1.1 or 1.2 - only DEA 1 strong positive or weak positive
DEA 1 breed predilection
Dal antigen
Breeds:
o Dalmatians - 12%
o Doberman Pinschers - 42%
o Shih-Tzus - 57%
o Dal negative is rare, most animals are Dal positive -> then we crossmatch a patient and it is incompatible with most of the donors - because it is Dal negative and donors are Dal positive.
Kai antigens
Canine blood typing
Canine crossmatching
Development of feline antibodies
o Not born with antibodies
Type A cats
Type B cats
Incompatible AB transfusions in cats
Mik antigen
Mik antigen naturally occurring antibodies
Feline inmunochromatographic test
What does plasminogen needs to be activated?
o tPA and fibrin and creates a complex
o Antifibrinolytic drugs (TXA and EACA) - lysin analogues, will bind to plasminogen and inhibit its binding to fibrin.
Cell base model diagram
Control of fibrinolysis
Vitamin K cycle
TRACS - Acute respiratory reactions - Pathophysiology of TRALI
o Is also based on the “two-hit” theory. First-hit risk factors include shock, positive intravascular fluid balance, hepatic surgery, low IL10 concentrations, systemic inflammation…. The increase in IL6, IL8 and C-reactive protein are seen with inflammation. C-reactive protein it is thought to contribute to the first hit by increasing concentrations of proteins that will activate and sequester neutrophils into the lungs. Neutrophils, monocytes, lymphocytes, platelets, neutrophil extracellular traps (NETS), the endothelium and other immune mechanisms might also play a role in the development of TRALI. In the two hit theory, primed neutrophils and endothelium are considered part of the first hit.
o The second-hit is due to the blood product transfusion by activating those neutrophils and endothelium, leading to pulmonary neutrophil infiltration and edema. The products with higher risk of causing TRALI are plasma and platelets, most likely because the bioactive mediators for TRALI lie within the plasma.
o These bioactive mediators can be immunologic, like antibodies to human leukocyte antigens, anti-HLA class I or II; various human neutrophil antigens, HNA - or non-immunologic like extracellular vesicles, aged blood cells, soluble CD40, among others. 80% of TRALI cases are immunologic, only about 20% are due to non-antibody factors.
TRACS - Infectious pathogens documented as capable to be transmitted by blood transfusion
These pathogens are documented:
I) to be transmitted (experimentally or clinically) by blood transfusion or by intravenous blood injection in canine and feline patients; and/or
II) to survive in stored blood units.
