Primary hemostasis (Proceedings)

Normal hemostasis occurs in three main stages. Primary hemostasis occurs when platelets attach to a damaged or disrupted area of the endothelium. This adhesion allows the platelets to undergo a shape change and then aggregate together. Once adhered to each other a temporary platelet plug is created. However, this platelet plug is not stable for long if the secondary hemostatic forces do not strengthen and reinforce the plug with a crosslinked fibrin mesh. The final stage of coagulation involves fibrinolysis. In this stage, plasminogen is activated to plasmin, which then breaks down the fibrin and removes the clot.

Primary Hemostasis

Primary hemostasis involves three main sequential steps. They are platelet adhesion, platelet activation, and platelet aggregation. Thus primary hemostasis involves platelets. Normal canine platelets have an average lifespan of approximately 6 days. Platelets are synthesized in bone marrow and smooth muscle cells, mainly in response to thrombopoietin. The platelet has no nucleus but does contain alpha and dense granules. Alpha granules contain substances such as platelet derived growth factor, fibronectin, transforming growth factor, platelet factor 4, fibrinogen, factors V and VIII, and von Willebrand factor (vWF). Dense granules contain adenosine diphosphate (ADP), adenosine triphosphate (ATP), histamine, epinephrine, serotonin, and calcium ions. These substances are released during platelet activation and maintain various roles. Some of these substances recruit other platelets to the site of injury.

Under physiologic conditions, hemostasis is prevented by the endothelium. This provides a physical barrier and secretes platelets inhibitory products, such as prostacycline (PGI2) and nitric oxide (NO). There are also substances expressed on the endothelial cell surface that degrade or inhibit platelet agonists. These include ADPases, heparin sulfate, and thrombomodulin.

Platelet Adhesion, Activation, and Aggregation

Platelet Adhesion

The first event in hemostasis is the adhesion of platelets to exposed subendothelium (ie collagen). In areas of high shear rate (in the microvasculature), this is mediated by von Willebrand factor (vWf), which binds to glycoprotein Ib-IX in the platelet membrane. In areas of low shear rate (e.g. aorta), fibrinogen mediates the binding of platelets to the subendothelium (by attaching to a platelet receptor - the integrin, glycoprotein Ia/IIa).

Platelet Activation

The adhesion of platelets to the vessel wall activates them, causing the platelets to change shape, to activate the collagen receptor on their surface (an integrin receptor called glycoprotein IIb/IIIa,now renamed Integrin aIIb 3) and to undergo the release reaction (release alpha and dense granule constituents). In addition, upon activation, platelets synthesize and release thromboxane A2 (TXA2) and platelet activating factor (PAF), which are potent platelet aggregating agonists and vasoconstrictors.

Platelet Aggregation

TXA2, PAF, ADP and serotonin are platelet agonists, causing the activation and recruitment of additional platelets, which bind to the adhered platelets. This activation is enhanced by the generation of thrombin through the coagulation cascade; thrombin being an important platelet agonist. Platelet aggregation is mediated primarily by fibrinogen (vWf has a secondary role), which binds to Integrin aIIb 3 on adjacent platelets. This aggregation leads to the formation of the primary platelet plug, which must be stabilized by the formation of fibrin.

Platelets also contribute to secondary hemostasis (coagulation cascade) by providing a phospholipid surface and receptors for the binding of coagulation factors. Another receptor that needs to be mentioned is the ADP receptor. Once ADP binds to its receptor, the Integrin aIIb 3 receptor undergoes activation thus facilitating platelet aggregation.

After the platelet plug has bridged the gap between endothelial cells, adjacent endothelial cells release prostacyclin causing vasodilatation and decreased platelet aggregation. This release of prostacyclin stops the platelet plug from growing out of control.

Disorders of Primary Hemostasis

Disorders of primary hemostasis can be separated into three categories: thrombocytopenia (ie low platelet numbers), thrombocytopathia (ie platelet function disorder), and vasculitis/endothelial disruption.

Thrombocytopenia can be caused by decreased production, increased destruction or sequestration. Decreased production can be caused by certain drugs (ie antibiotics), infections, myelodysplasia, or it can be idiopathic. Platelet destruction is usually a result of immune mediated diseases, but can also be caused by infections or neoplasia. Sequestration of platelets occurs with splenic disease or during endotoxemia. Heparin induced thrombocytopenia (HIT) is a common primary hemostatic disorder in humans. There are two forms of HIT recognized. Type I HIT is a non immunologic disorder where heparin binds directly to the platelet surface. Platelet numbers are decreased due to increased clearance of this abnormal platelet. Type II HIT is an immunologically mediated process in which antibodies to heparin bind to the heparin-platelet complex and destruction occurs. These heparin-platelet-antibody complexes are also capable of forming thrombi.

Thrombocytopathia can be an acquired or inherited condition. Acquired thrombocytopathia can be due to certain infections (ie Ehrlichiosis, feline leukemia virus), drugs (ie aspirin), liver disease, uremia, or neoplasia. Inherited thrombocytopathia is recognized most commonly as von Willebrand's disease (vWD). vWD is thought to be the most common inherited primary hemostatic disorder in dogs. As discussed previously, vWF has two main roles-1. it is a carrier protein for FVIII and 2. it mediates platelet adhesion to the damaged endothelium. vWD has three types. Type 1 vWD is where vWF multimers are decreased in number. Type 2 vWD is where the large multimers are absent (and the large multimers aggregate platelets to much greater degree). Type 3 vWD is where no multimers are present.

Vasculitis or endothelial destruction can cause primary hemostatic disorders as well. Such disruption of the endothelium can cause problems with platelet numbers or function due to lack of platelet receptors.

Inhibitors of Primary Hemostasis

With advancing knowledge about the coagulation cascade (ie both primary and secondary hemostasis) and its role in several disease states, novel therapies have been developed to combat clot formation. Inhibition of primary hemostasis has gained interest as multimodal therapy has been shown to be more effective. There are three main categories of drugs that inhibit primary hemostasis.

Thromboxane Inhibitors

Aspirin is the classic thromboxane inhibitor. It irreversibly inhibits the cyclooxygenase enzyme in platelets and endothelial cells. The cyclooxygenase enzyme is responsible for converting arachidonic acid into endoperoxidases, which are then either converted to thromboxane or prostacyclin via thromboxane synthetase or prostacyclin synthetase respectively. In platelets, aspirin irreversibly inhibits cyclooxygenase through acteylation of a serine residue of key importance for the function of the cyclooxygenase system. This acetylation causes inhibition of TXA2 and serotonin. Normal function is restored 5-10 days after a single dose of aspirin. Since new platelets are produced daily there is a gradual increase in function over this period of time. In endothelial cells, aspirin has a different role. Although it still inhibits the cyclooxygenase enzyme, these cells favor prostacyclin production over thromboxane production. As stated previously, prostacyclin is actually a platelet inhibitor (ie it inhibits platelet aggregation). For years there has been much debate over aspirins' true effect. If it inhibits prostacyclin how can it be anti-thrombotic? Much research has been conducted in this area. The answer lies in the fact that platelets have no nucleus while endothelial cells do. Therefore, endothelial cells can produce more cyclooxygenase enzyme within a matter of hours. It has been suggested that by giving low dose aspirin (1mg/kg PO q 24 hours) you would inhibit platelets but allow endothelial cells to continue release of prostacyclin. Although other thromboxane inhibitors have been developed, aspirin remains the most effective agent.

Glycoprotein IIb/IIIa (now called Integrin aIIb 3) Receptor Inhibitors

As mentioned earlier, the Integrin αIIb β3 is essential during platelet aggregation. Several drugs have been formulated to block the integrin receptor thus inhibiting platelet aggregation. Such drugs include Eptifibatide, Abciximab, and Tirofiban. These drugs are all injectable as the oral preparations have not been found to be effective. Although these drugs are not commonly used clinically in veterinary medicine, one research group (JM Bright et al) did publish their findings with the use of abciximab in cats. They found that although abciximab plus aspirin did not inhibit thrombus formation completely, it did slow the formation compared to aspirin therapy alone.

ADP Receptor Antagonists

As mentioned earlier, ADP and the binding to its receptor are important during platelet aggregation. The thienopyridines, clopidogrel ("Plavix") and ticlopidine, have been formulated to antagonize the ADP receptor thus inhibiting platelet aggregation. The use of Plavix in cats with heart disease to prevent clot formation is slowly becoming standard practice. A recent study by Hogan et al looked at various doses of Plavix for preventing platelet aggregation in cats. They found that 18.75mg (ie ¼ of the standard 75mg tablet) once a day was effective at preventing platelet aggregation. They thus theorized that this dose could be used to prevent aortic thromboembolism.

References

McMichael M. Primary Hemostasis. JVECC 15(1) 2005, pp 1-8.

Roberts HR, Monroe DM, Escobar. Current Concepts in Hemostasis. Anesthesiology 100 (3) 2004 pp 722-30.