Hemostasis, the process of blood clot formation, is a series of coordinated responses to vessel injury. The coordinated activity involves activation of platelets, the coagulation cascade, blood flow and shear, endothelial cells, and fibrinolysis. Too little hemostasis results in hemorrhage. Too much hemostasis results in thrombosis.

Platelet plug formation

Physiological activation of platelets at the site of vascular injury include adenosine diphosphate (ADP), epinephrine, thrombin, and collagen. Thrombin and collagen are strong agonists. Thrombin is mediated by G protein-coupled protease-activation receptors (PAR). There are also specific receptors for the other agonists. Platelet activation involves four distinct processes: adhesion (deposition of platelets on subendothelial matrix); aggregation (cohesion of platelets, secretion (release of platelet granule proteins); and procoagulant activity (enhancement of thrombin).

Adhesion-Platelet adhesion is primarily mediated by the binding of platelet receptor glycoprotein (GP) complex (Ib-IX-V) to the adhesive protein von Willebrand factor (vWf) in the subendothelial matrix. Deficiency of the complex (Bernard-Soulier disease) or of vWf (von Willebrand disease-vWd) can be clinically significant.

Aggregation-Platelet aggregation involves binding of fibrinogen to the platelet fibrinogen receptor (GPIIb-IIIa complex also called integrin alpha IIb beta 3). Integrins make up a superfamily of adhesive GPs. Congenital deficiency of GPIIb-IIIa leads to Glanzmann thrombasthenia and afibrinogenemia.

Protein secretion-After activation platelets secrete ADP and serotonin which, in turn, stimulate and recruit other platelets. Adhesive proteins such as fibronectin and thrombospondin reinforce and stabilize platelet aggregates; coagulation protein V is involved with the coagulation cascade; thromboxane stimulates vascular constriction; and platelet derived growth factor (PDGF) stimulates proliferation of smooth muscle cells and mediate tissue repair.

Endothelial cells-It is likely that endothelium had distinctive subsets depending on the arterial, venous or microcapillary areas. This is an area which is likely to provide some clinical insights as to the proclivity of thrombosis in a given patient.

Procoagulation-Platelet activation also results in the assembly of the coagulation proteins (enzymes) on the platelet surfaces.

Coagulation-The Clotting Cascade. The classic views of coagulation-the intrinsic, extrinsic and common pathways-while convenient, is not completely accurate. Patients who are severely deficient in factor XII (perhaps many cats), or even factor XI do not clinically bleed which suggests the contact phase of coagulation is not critical in vivo. The exposure of tissue factor (factor III) at the wound site is required to activate factor VII and, thus, to initiate clotting either directly by activating factor X or indirectly by activating factor IX.

All of the procoagulants are synthesized in the liver except vWf which is synthesized by megakaryocytes and endothelial cells. The vitamin K dependent carboxylation of glutamic acid residues acts as a recognition signal that guides the posttranslational modification of the proteins for biologic activity.

Interaction between activated platelets and coagulation

Activated platelet expose anionic lipids on the platelet surface. Factor V, stored in platelets, is activated, released and bound to these lipids forming the assembly site for activated factor X and prothrombin (factor II). The platelet plug keeps thrombin localized. Similar activation occurs with factor X (when bound to factor V is protected from antithrombin III [ATIII] inactivation) activated factors VIII and IX.

Control mechanisms-Coagulation (secondary hemostasis) is modulated by dilution of procoagulants, removal of activated factors by the mononuclear phagocyte system (MPS), and control by inhibitors of thrombosis by ATIII, protein C, protein S, tissue factor pathway inhibitor (TFPI) which collectively regulate coagulation, prostacyclin (PGI) and nitric oxide which regulate vascular and platelet activity, and by fibrinolysis which removes the fibrin clot. .

Fibrinolysis-Tissue plasminogen activator (t-PA) is released from damaged endothelial cells and converts plasminogen to plasmin. This optimally occurs on a surface somewhat analogous to ATIII and heparin sulfate. Plasmin cleaves polymerized fibrin and fibrinogen at multiple sites releasing fibrin and fibrinogen degradation products (FDPs) including D-dimer. Plasmin is inactivated by alpha 2 antiplasmin. Urokinase is the second physiologic plasminogen activator found in high concentrations in urine and is the major fibrinolysin in the extravascular area. The major inhibitors of plasmin and urokinase are plasminogen activator inhibitor-1 (PAI-1). There are other fibrinolysis inhibitors such as thrombin activatible fibrinolysis inhibitor which have not been well studied.

Overview of Hemostasis

Hemostasis is initiated by the exposure of tissue factor at the wound site resulting in thrombin generation and deposition of fibrin. Simultaneously, damaged endothelium relaeases t-PA, production of plasmin and clot lysis. Both thrombin and plasmin are under control of their respective inhibitors, ATIII and alpha 2 antiplasmin. When there is coordinated symmetry, the clot prevents bleeding, followed by clot lysis and tissue remodeling. Diminished thrombin formation and increased plasmin formation results in hemorrhage. The converse is equally probable and depends on the inciting process and the physiologic status of the patient.

Diagnostic tests and their use

Tests of platelets and platelet function-primary hemostasis

Peripheral blood smear evaluation should reveal 8-12 platelets per 1000 x magnification corresponding to 150,000 to 300,000 platelets per microliter. The smear also is useful in determining platelet granularity and the presence of abnormally sized and shaped platelets. The buccal mucosal bleeding time (BMBT) is used in patients with suspected problems with primary hemostasis (endothelial cells and/or platelets) but with appropriate platelet counts. It is useful in screening patients for vWd or certain other platelet function disorders.

Platelet aggregometry utilizes photometric devices to detect the clumping of platelets-increased light passing through the platelet rich test tube. Various concentrations of platelet agonists such as ADP cause a primary wave of aggregation followed by a secondary wave. the secondary wave reflects the induction of platelet release of granule contents. A subnormal secondary wave indicates platelet storage pool defects or diminished release activity. The former is usually indicative of congenital platelet diseases and the latter is associated with drug effects.

Tests of the coagulation cascade-secondary hemostasis

Most coagulation tests measure the time for fibrin production. Prolongation may represent low factor concentration, inactive factor or factors, or the presence of inhibitors. The APTT examines the intrinsic and common paths but is most sensitive to dysfunction in the intrinsic path. The PT is a test of the extrinsic and common paths and is now being reported using the international normalized ratio (INR = [log patient PT/log control PT]c) where c represents the international sensitivity index (ISI). In this way the thromboplastin used in an individual laboratory is calibrated against a standard reference thromboplastin and the PT is reported as an INR. The thrombin time (TT) is used to test abnormalities of the conversion of fibrinogen to fibrin and can be prolonged because of hypofibrinogenemia, dysfibrinogenemia or the presence of inhibitors such as FDPs. Fibrinogen can be measured antigenically or more commonly by clotting assays and is reported in mg/dL. FDPs result from degradation of fibrinogen and fibrin. D-dimer results from plasmin-mediated degradation of fully polymerized fibrin and is a specific measure of intravascular fibrin deposition and plasmin degradation characteristic of disseminated intravascular coagulation (DIC).

Tests of inhibitors of hemostasis-Mixing studies

A prolonged APTT can be caused either by a coagulation factor deficiency or an inhibitor generated against a specific coagulation protein or a phospholipid-protein complex-the so called lupus anticoagulant. Mixing one volume of pooled plasma and one volume of patient plasma should correct a prolonged APTT resulting from a coagulation deficiency but not due to an inhibitor. ATIII, Protein C, Protein S - A functional assay is best when examining ATIII or protein C or protein S concentrations.

REFERENCES

References are available on request.