Hemostasis Flashcards
Occurrence of Hemostasis
A regular process that is continuously occurring due to minor damages to vasculature
Balance between the procoagulants and anticoagulants allowing clot formation and lysis
- Normally more anticoagulants than procoagulants to prevent unwanted clots from forming
Procoagulants vs. anticoagulants
Procoagulants: promote coagulates
Anticoagulants: inhibit coagulants
More than 50 different substances involved in coagulation
Hemostasis
- A complex process involving proteins, cells, and blood vessels to stop bleeding
- Involves primary and secondary hemostasis
What activates hemostasis?
Procoagulants
Activation of hemostasis
- Platelet plug forms
- Thrombin forms fibrin clot and inhibits fibrinolysis
Activation of fibrinolysis
- Stimulation for activation decreased or absent
- Thrombin inhibits own production and no longer inhibits fibrinolysis
Disorders involving excessive clotting
- Deep vein thrombosis (DVT)
- Stroke
- Myocardial infarction
- Embolism
- Disseminated intravascular coagulation (DIC)- occurs right before death. Usually something else causing it. Need to find it quickly!
Disorder involving inadequate clotting
hemorrhage
Primary Hemostasis
- vascular constriction/vasospasm
- lasts minutes to hours, more severe with greater trauma - Platelet plug formation
What causes the vascular spasm in primary hemostasis?
- Local myogenic spasm (smooth muscles fibres contract due to local factors released from surrounding tissues)
- Local factors from traumatized tissues, vascular endothelium, and platelets
- Nervous reflexes initiated by pain nerve impulses
Platelet plug formation events
- Platelet capture
- Platelet adhesion
- Platelet activation
- Platelet plug formation
Platelet capture and adhesion
- Injury site exposes adhesive proteins underneath (vWf and collagen or laminin, fibronectin)
- Inactive platelets with integrin and non-integrin receptors that can bind to the adhesive proteins
- Decision of which receptors and proteins binds depends on blood flow. In areas of high flow, Von Willebrand factor (vWf) must bind first but in slower blood flow rates, can bind collagen first.
Agonists in platelet capture
Thrombin can cause the release of Weibel-Palade bodies (intracellular stores of Von Willebrand factor (vWf)) from endothelial cells which supplies more adhesive proteins to the area, promoting platelet capture and adhesion
Von Willebrand Disease (vWD)
- Lack of Von Willebrand factors (vWf)
- Identified in many dog breeds but rare in other domestic species
- Individuals will often go undetected until they have a surgery or injury that tips the balance
Platelet Activation after adhesion
When platelets bind, they do a series of things to help prepare for secondary hemostastis. Need calcium.
- Change shape
- Release granule content and calcium
- Membrane metabolism
- Membrane flipping
- Release of procoagulant microparticles
- Activate fibrinogen receptors to bind fibrinogens
These steps prepare everything for platelet aggregation.
Platelet membrane metabolism
Synthesize eicosanoids such as thromboxane A2 which are platelet agonists
Platelet membrane flipping
- Phosphatidylserine from inner to outer layer
- Allows for clotting factors to assemble and bind (clotting cascade preparation)
Platelet release of procoagulant microparticles
Increase surface area for fibrin formation
Platelet activation of fibrinogen receptors to bind fibrinogens
Prepares for secondary hemostasis by providing receptors to bind fibrinogen which is needed for platelet aggregation and therefore a clot
Calcium and Platelet activation/aggregation
- Needed for all steps that allow for preparation for secondary hemostasis/clotting cascade.
- Removing calcium prevents clotting
Platelet Aggregation
- Activation steps set the stage by attracting more platelets, increasing SA, assembling clotting factors, and providing receptors.
- Fibrinogen connect platelets together binding to receptors forming primary platelet plug.
Cells involved in platelet plug formation
- Platelets
- Endothelial cells
Proteins involved in platelet plug formation
On injured site:
- Von Willebrand factor (vWf)
- Subendothelial matrix proteins (eg. Collagen)
On plasma membrane of platelets
- Integrin receptors
- Non-integrin receptors
What activates formation of platelet plug?
- Thrombin
- Collagen
- Thromboxane
- ADP
What inhibits formation of platelet plug?
- Nitric oxide
- Prostacyclin (PGI2)
- ADPase (made by endothelial cells)
Megakaryopoiesis
Production of megakaryocytes
Thrombopoiesis
- Production of platelets from megakaryocytes
- Occurs mostly in the bone marrow but 7-15% occurs in the spleen and lung
Platelet production
- Endomitosis- division of chromosomes without nuclear division. Results in mature megakaryocytes.
- Mature megakaryocytes are polypoid (8N-64N)
- 1 Megakaryocyte can produce 1000-5000 platelets
What regulates megakaryocytes and platelet production?
- Thrombopoietin (TPO)
- Produced mainly in liver
Feedback mechanisms in platelet regulation
Thrombopoietin (TPO) concentration is inversely proportional to platelet concentration
- Platelets continuously sequester TPO to maintain normal levels of platelets in circulation
Thrombocytopenia leads to decreased sequestration of TPO into platelets
Key points of CBC for platelets
- Thrombocytopenia (decreased number)
Caused by:
- Decreases bone marrow production (drugs or genetic factors)
- Increased consumption (coagulation)
- Increased clearance (macrophages getting rid of them) - Thrombocytosis (increased number)
Caused by:
- Drug induced (eg. corticosteroids)
- Disease-associated (eg. Cancer) - Clumping or abnormal morphology
Secondary Hemostasis
Formation of the blood clot through a complex cascade of chemical reactions
Fibrinogen to fibrin reaction
- Thrombin converts fibrinogen into fibrin strands
- Fibrin-stabilizing Factors (XIII) cross links fibrin strands and stabilizes the clot
Where are most coagulation factors synthesized?
- Liver
- Activated through cloting cascades
Intrinsic vs. extrinsic factors
- Intrinsic- blood clotting inside the body (internal damage)
- Extrinsic- blood clotting outside the body (external trauma/damage), faster
Key components of blood clot formation (secondary hemostasis)
- Coagulation factors
- Enzymatic coagulation factors (enzymes)
- Non-enzymatic coagulation factors (co-factors)
- Fibrinogen substrate
- Calcium - Platelet phospholipid surfaces
Importance of platelet phospholipid surfaces
Provide the platform for coagulation cascade reactions
Steps of coagulation process
- Initiation of thrombin generation
- Amplification of thrombin generation
- Propagation of thrombin generation
- Fibrin formation
Initiation of thrombin generation step
- Damage to endothelium which exposes Tissue factor (TF) on fibroblasts
- TF forms a complex with Factor VII initiated a cascade
- Activates Factor X which works with co-factor V which then activates change of prothrombin to thrombin (small amount)
Amplification of thrombin generation step
Small amount of produced thrombin (from prothrombin) will activate the membrane surface of platelets to provide a scaffold for the other factors to be activated
- Membrane surface will be flipped outwards to display phosphatidylserine (PS) which has chemistry that allows clotting factors to bind to the platelet surface
Propagation of thrombin generation step
- Activated platelets provide a critical surface for the assembly of coagulation complexes, essential for clot expansion
- Activation of a large amount of factor X leads to a large amount of thrombin being made from prothrombin
- Thrombin activates fibrinogen to make fibrin
Fibrin formation step
- Thrombin activates fibrinogen, making fibrin
- Fibrin strands will crosslink together forming the clot (completed by Fibrin-stabilizing Factors (XIII))
Roles of antithrombin and fibrin
Antithrombin
- Binds to free thrombin in the circulation
- Prevents new clot propagation beyond the injury site
Fibrin
- Binds thrombin, reducing its availability to convert more fibrinogen
- Localizes the clotting process to the site of vascular injury
Balance between hemostasis and fibrinolysis
At site of injury, both processes immediately begin however clotting is greater than fibrinolysis at the start where inactivated plasminogen just gets trapped within the clot. Then gradually switch occurs
Fibrinolysis
- The breakdown of blood clots
Plasminogen (inactive) –> plasmin –> degrades fibrin fibers, procoagulants (fibrinogen, prothrombin, factors V, VIII, XII)
- Process activates by tissue plasminogen activator (t-PA) or Urokinase
- Process inhibited by plasminogen activator inhibitor (PAI) or alpha2-antiplasmin
Activators of fibrinolysis
tissue plasminogen activator (t-PA) or Urokinase
Deactivators of fibrinolysis
plasminogen activator inhibitor (PAI) or alpha2-antiplasmin
Steps of fibrinolysis
- Inactivated plasminogen is trapped within the clot
- Endothelial cells near injury site will release both fibrinolysis activators (tissue plasminogen activator (t-PA) or Urokinase) and deactivator (plasminogen activator inhibitor (PAI))
- When activators override the deactivators, plasminogen will start to become activated, becoming plasmin
- Plasmin degrades clots into fibrin degradation products
Alternative way that fibrinolysis can occur
Another way that fibrinolysis can occur is by the alpha2-antiplasmin (produced in liver or by platelets) circulating body. Will bind to active plasmin and prevent too much fibrinolysis from happening.
Intravascular anticoagulants
Prevent clotting in the normal vascular system
- Antithrombin inhibits free thrombin and factor X
- Heparin (produced by mast cells and basophils) amplifies antithrombin 100-1000x when combined
Anticlotting in the normal endothelium
- Vessel wall smoothness prevents unintended clotting trigger
- Glycocalyx barrier repels unneeded clotting factors and platelets
- Endothelial cells produce thrombin inhibitors and vasodilators like nitric oxide and prostacyclin
Drugs/toxins that affect clotting cascade
- Warfarin and dicoumarol
- Factor X inhibitors
- Heparin
Warfarin and dicoumarol
- Antagonists of vitamin K which results in inhibition of Factors VII, IX, X and prothrombin and inhibits coagulation
- Delayed anti-coagulant effect
Factor X inhibitors
- Most commonly used oral anticoagulants
- Inhibits factor X and therefore Thrombin production
Heparin
- Produced endogenously at very low levels by mast cells and basophils, and also used as a drug
- Causes enhanced activity of antithrombin which in turn inhibits Factor X and thrombin
- Used in blood tubes
Importance of vitamin K and blood clotting
- Vitamin K is required for the carboxylation of several clotting factors which is needed for the factors to bind calcium ions
- Calcium binding allows for clotting factor to bind to plasma membrane of platelets
Conditions that affect primary hemostasis
- Thrombocytopenia- due to decreased production or increased destruction of platelets
- Von Willebrand disease- common in dogs; lack of vW factors which are needed for trapping and adhesion of platelets to injury site
What clinical symptoms would be displayed when primary hemostasis is affected?
Superficial hemorrhages involving skin and mucosal surfaces
- Petechia- minor tearing of blood vessels
- Hematochezia- bloody stool
- Hematuria- bloody urine
- Epistaxis- bloody nose
Conditions that affect secondary hemostasis
- Liver diseases (hepatitis, cirrhosis)- impair production of blood-clotting factors
- Vitamin K deficiency (diet or GI disease that impair absorption of vitamin K)- needed for clotting factors
- Congenital disorders
Clinical symptoms of impairment of hemostasis
Larger, wide spread bleeding
Eg. Hematomas, bleeding in body cavities
Disseminated intravascular coagulation and Fibrinolysis (DIC &F)
- Death is coming, and Fast
- Simultaneously bleeding and clotting excessively
- Severe condition that occurs secondary to another underlying issue (sepsis, trauma)
Common laboratory evaluation for primary hemostasis
- CBC
- Buccal mucosal bleeding time (BMBT)- how long it takes for a platelet plug to form after a superficial vessel is cut
- Von Willebrands factor assay- measured by ELISA
Common lab evaluation for secondary hemostasis
- CBC
- Activated coagulation time (ACT) – evaluate the function of the intrinsic pathway
- Partial thromboplastin time (PTT)- test of the intrinsic and common pathway function
- Prothrombin time (PT)- test of the extrinsic and common pathway (most often used to assess therapy for warfarin toxicity)
