Lecture 14a: Hemostasis

HEMOSTASIS

Hemostasis is the process which retains fluid blood within the vascular system in spite of the injury to the vessel wall. It is the entire mechanism by which bleeding from an injured blood vessel is controlled and finally stopped. The process is a progression or chain of physical and biochemical changes normally initiated by injury to the tissues and blood vessels and culminating in the transformation of fluid blood into a solid thrombus or clot which effectively seals the torn vessels. The entire mechanism is divisible into three aspects:

A. Extravascular phenomena

These consist of:

1. The physical effect of the surrounding tissue, e.g., skin, muscle, elastic tissue, in tending to close the seal the rent in the injured blood vessel.

2. The biochemical effect of substances released from the injured tissue and reacting with plasma and platelet factor.

B. Vascular phenomena

The damaged blood vessel constricts almost instantaneously. This process is known as vasoconstriction and is important in the early control of hemorrhage following injury to a vessel. However, this reflex nervous vasoconstriction tends to pass of within a relatively short, though variable time but it is possibly enhanced by local release of substance, serotonin. Serotonin is released from platelets as these adhere or stick to the margins of the injured or defect in all of the vessel. It promotes a local, direct, biochemically stimulated narrowing of the torn blood vessel and of intact vessels in the immediate vicinity.

C. Intravascular phenomena

These consist of the enormously complex sequence of physicochemical reactions which transform fluid blood into a firm fibrin clot and involve various factors.

In circulating blood there appears to be a balance between the forces which act to stimulate the formation of thrombin and the forces which tend to delay its formation or inhibit its action. This balance maintains blood in its fluid state.

This balance may be shifted either toward delayed thrombin formation or toward excessive clot formation.

THEORIES OF BLOOD COAGULATION

Many hypotheses have been proposed in an attempt to explain the sequence of events that leads to the development of a fibrin clot. Each hypothesis has been helpful by serving as a stimulus to investigators to test the concept. A hypothesis may remain popular because it is a true explanation of a natural phenomenon or because there is no way to design experiments to test its validity.

The sequence of events resulting in the conversion of prothrombin to thrombin has been visualized as a waterfall by Dacie and Ratnoff or as a cascade by McFarlane. In 1905 – 1906, P. Morowitz published his theory of blood coagulation. This theory provided a satisfactory basic working hypothesis which remained more or less unchanged for years. Morawitz theory has been proven to be basically correct. However, recent advances have considered the thromboplastin generation and thromboplastin activity and from this additional information, came the modern concept of blood coagulation.

Aside from those mentioned, other investigators of the clotting mechanism who published their own hypotheses were Quick and Howell.

SYNOPSIS OF MODERN CONCEPT OF BLOOD COAGULATION

Stage I:          Generation of Thromboplastin Activity

Here, two initially separate mechanisms are involved: the extrinsic thromboplastin generating mechanism. While these two mechanisms are initially separate, they progress through the same final pathway to produce the definitive thromboplastin, which in turn, causes the conversion of prothrombin to thrombin to the common end result, conversion of fibrinogen to fibrin, i.e., clotting.

        Extrinsic (Tissue) Thromboplastin Generating Mechanism

All injured tissues yield a complex mixture of substances both heat labile and heat stable which possess potential thromboplastic activity. These “tissue thromboplastic” substances are activated by coming into contact with plasma, specifically with factor VII of the plasma.

Intrinsic (Blood) Thromboplastin Generating Mechanism

This is an exceedingly complex system involving the interaction of several factors. Most are associated with the globulin fraction of blood proteins and are present in exceedingly minute amounts.

The Common Pathway

The common pathway of coagulation begins with the activation of Factor X. This step is calcium dependent and involves proenzymes to enzyme transformation which is accomplished by the two different enzymatic activities that evolve from the reactions of the intrinsic and extrinsic pathways. The interaction of Factor Xa with Factor V, calcium ions and Platelet Factor 3 leads to the activation of prothrombin.

Stage II:         Conversion of Prothrombin to Thrombin

The conversion of prothrombin into thrombin is calcium dependent and when studied in whole blood in vitro, is relatively slow but usually complete, i.e., all the prothrombin normally is converted into thrombin.

Stage III:        Conversion of Fibrinogen into Fibrin Clot

The last phase of coagulation involves the conversion of fibrinogen into stabilized fibrin and occurs in three steps:

a. The enzymatic step – thrombin is a potent proteolytic enzyme and its proteolytic action on fibrinogen occurs normally in the absence of calcium and releases 4 fibrinopeptides per more of fibrinogen. The residual molecule is termed monomer.

b. The polymerization step – this step leads to the formation of soluble fibrin. As a consequence, soluble fibrin is mechanically fragile and dissociates into its constituents monomers in the presence of inhibitors of hydrogen bonding, such as urea and monochloroacetic acid.

c. The stabilization step – the soluble fibrin is converted into insoluble or stabilized fibrin by Factor XIIIa. Insoluble fibrin provides an extremely strong and stable framework for the permanent hemostatic plug.

Miscellaneous Coagulation Phenomena

1. Role of Metal ion

Calcium ions are required for all reactions in the coagulation phase except those of the contact phase involving Factor XII and XI and the enzymatic effects of thrombin on fibrinogen and Factor XIII. Calcium ions apparently act as non – specific accelerators of fibrin polymerization but are not an absolute requirement for this step.

2. Consumption of Coagulation Factors

The coagulation factors have long been separated into two groups on the basis of their presence or absence in serum, i.e., those that are utilized or consumed during in vitro coagulation (fibrinogen, prothrombin, Factor V, VIII and XIII) and those that are not (Factor VIII, IX, XI, X, XII).

3. Initiating reactions

The coagulation system appears to be initiated by the activation of Factor XII and/or Factor VII. It is still unclear what the initial stimuli for activation of these factors are in vivo. Once formed, activated Factor XII (XIIa) participates in a positive feedback loop involving prekallikrein and high molecular weight kinninogen (HMWK) to generate more XIIa. Surface contact and the presence of HMWK also greatly facilitate the activation of Factor XI and XIIa.

4. The Contact Phase

Factor XII, Factor XI, prekallikrein and HMWK are plasma proteins involved in the earliest stages of blood coagulation. Because of their activation by glass and other foreign surfaces, they have been referred to as contact factors. Biologic surfaces that produce contact activation include collagen fibers, unbroken skin, sebum, long chain fatty acid, uric acid, homocysteine and possibly elastin, and they are then called surface contact activators.

5. The role of tissue factors

The tissue play an important role in hemostasis for the elasticity of the skin and mucous membrane automatically tends to close wounds. When tissues are traumatized or blood vessels and lymphatic channels are disrupted or cut, fluids from the injured cells and from the tissue spaces escape. This fluid contains a potent clot–activating substance known as tissue thromboplastin (Factor III). Traumatized WBC, RBC and platelets contribute to the tissue thromboplastin complex.

6. Role of platelet in coagulation and hemostasis

a. Platelet adhesion – platelet adhere to collagen and microfibrils present in vascular basement membrane. Platelet adhesion to collagen is dependent on the degree of polymerization of collagen and presence of free epsilon – amino group or collagen.

b. Platelet aggregation – platelet aggregation indicates platelet–to–platelet adhesion. The formation of platelet clumps occur normally in vivo following vascular injury. The platelet aggregates mechanically block the outflow of blood from injured blood vessels.

c. Action of platelets on fibrin formation and clot retraction

(1)   Platelet supply platelet factor 2 which accelerates the conversion of fibrinogen to fibrin

(2)   Platelet membranes provide a phospholipid, platelet factor–3 which catalyzes the activity of certain calcium dependent procoagulant interactions. Platelet factor–3 accelerates the interaction of coagulation factors IXa, VII and calcium in the activation of Factor X; PF 3 also aids in the interaction of Xa, V and calcium in the activation of prothrombin.

(3)   Platelet factor 4 (PF4) has anti–heparin–like activity and is liberated during release. PF4, therefore, prevents inhibition of coagulation in and around a platelet clot.

(4)   Clot retraction is due to the action of actomyosin (thrombosthenin), the platelet contractile protein

(5)   Procoagulant activity of platelets – serves as a source of thromboplastin in native clotting system.

NOMENCLATURE FOR COAGULATION FACTORS

Circulating procoagulants have been given descriptive names (fibrinogen, prothrombin, thrombin), functional names (labile and stable factors), and surnames of the kindreds in whom hereditary defects were first discovered (Hagemen, Fletcher and Stuart factors). In order to avoid confusion, an International Committee established a nomenclature of blood clotting factors. Roman numerals were used to denote factors in order of their discovery without regard to their position in the sequence of the reactions. In addition, Factor VI was initially the activated form of Factor V. The term has since been dropped. Factor IV is not a circulating procoagulant but represents calcium ions. The non – activated factors have been assigned a Roman numeral and the activated forms are indicated by an appended “a.” Factors V and VII may not have activated forms.

PHYSICAL AND CHEMICAL PROPERTIES OF PROCOAGULANTS

Factor I – Fibrinogen

Fibrinogen is a plasma protein which is formed in the liver and is converted into fibrin in the presence of thrombin. Approximately, 75% of the total body pool of this is present in the plasma where the concentration ranges from 250 – 500 mg/dl. It is absent in the serum and remains unchanged in absorbed plasma. It is precipitated by 23 – 45% ammonium sulfate.

Factor II – Prothrombin

Prothrombin is a proenzyme, the precursor of thrombin. It is precipitated by 50% ammonium sulfate and is present in human plasma in concentration of approximately 10 – 20 mg/dl. Vitamin K is essential for the production of prothrombin by the liver and probably in the lungs. It is consumed in the clotting process and normal serum contains only a trace amount of it (residual prothrombin). Prothrombin is also related to Factor VII which is also manufactured in the liver, indeed there is evidence that Factor VII can be converted to prothrombin by the liver.

Factor III – Tissue Thromboplastin

The term thromboplastin was used to designate the clot accelerating action of extracts of tissues and this term accelerating activity of tissues has been assigned the term Factor III.

Complete tissue thromboplastin is used to designate active tissue thromboplastin preparations which are able to produce clotting as rapidly with hemophilic plasma as with normal plasma.

Partial tissue thromboplastin is used to designate those reagents or preparations that are found to clot hemophilic plasma less rapidly than normal plasma.

During clotting of whole blood, platelets appear to be the source of thromboplastin. Thromboplastin is required for the conversion of prothrombin to thrombin.

Factor IV – Calcium Ion

Calcium is present in normal blood from 9 to 11 mg/dl. Half of this is ionized and since only a very small amount of ionized calcium is required in the clotting mechanism, defects which result from calcium deficiency are not seen except during massive transfusion with citrated blood.

Factor V – Proaccelerin

Factor V is synthesized in the liver. It is unstable when stored in citrated plasma and is rapidly inactivated in vitro by strong chelating agents. It is consumed during clotting process and is not found in the serum. It is not affected by Dicumarol and Vitamin K administration. It is inactivated by heating to 58^oC and is precipitated by 33% ammonium sulfate. The in vitro activity of Factor V is increased by the action of thrombin, Russell’s viper venom and papain.

Factor VII – Serum Prothrombin Conversion Accelerator

This factor functions together with thromboplastin to hasten prothrombin conversion. Its activity in serum is increased but absent in adsorbed plasma. This is related to prothrombin and is capable of being converted to prothrombin by the liver which makes both Factor II and VII subject to the influence of Vitamin K. It is not destroyed during the clotting process and is present in both serum and plasma. Dicumarol administration produces a reduction in this factor.

Factor VIII – Antihemophilic Factor

This factor is consumed during the clotting process and therefore is not present in the serum. It is precipitated by 25% ammonium sulfate. It is unstable in citrated plasma and is rapidly inactivated by strong chelating agents. Deficiency in this factor is the cause of hemophilia.

Factor IX – Plasma Thromboplastin Component

This factor is present in both plasma and serum. It is Vitamin K dependent and is adsorbed by aluminum hydroxide and barium sulfate. It is an essential component of the intrinsic thromboplastin generating system.

Factor X – Stuart–Prower Factor

This factor is not consumed in the clotting process and is found in both serum and plasma. It is precipitated by 55 – 65% ammonium sulfate. It is activated by the products of both the intrinsic and extrinsic systems.

Factor XI – Plasma Thromboplastin Antecedent

This is beta and gamma globulin which is partly consumed in the clotting process and is therefore found in both serum and plasma. It is precipitated by 50% ammonium sulfate.

Factor XII – Hageman Factor

This factor is activated by contact with foreign surface and initiates the intrinsic system. It is also involved in the activation of fibrinolysis

Factor XIII – Fibrin Stabilizing Factor

This protein the linkages between the fibrin monomers of the blood clot. It is present in the plasma, platelets and apparently synthesized by megakaryocytes.

Prekallikrein – Fletcher Factor

This is a plasma protein with a molecular weight of approximately 100,000 daltons. It is partially adsorbed by barium sulfate but not adsorbed by aluminum hydroxide.

High Molecular Weight Kininogen–Fritzgerald Factor

This factor is a protein in normal plasma with a molecular weight 200,000 daltons and migrates as alpha globulin.

Coagulation, therefore, may be defined as the process whereby blood is changed from liquid state to that of soft jelly like solid. After the formation of a solid clot, a clear yellowish liquid exudes from the clot. This liquid is known as the serum and the process of its separation from the clot is called clot retraction. This process is believed to be the function of the platelets. The subsequent process to take place is the lysis or dissolution of the clot, which is termed fibrinolysis.