Blood CoagulationZHemostasis

The term blood coagulation or hemostasis can be defined as a cascade of enzyme activation which enables the stoppage of bleeding from injured vessels, help to keep the blood in fluid state during circulation and to resolve the clot for restoring vascular integrity.

4.4.1 The Coagulation Machinery

The process of blood coagulation and its associated clot retraction and fibrinolytic (degradation of fibrin clot) mecha­nism is the result of complex interactions of some soluble clotting factors as well as platelets and vascular endothelium. The structure and the development of platelets were discussed in previous chapters. Here, we will mainly focus on the role of platelets in blood coagulation.

The Clotting Factors/Coagulation Factors The clotting factors/coagulation factors involved in hemostasis are listed in Table 4.17. Most of the clotting factors are produced in the liver except Factor-III (plasma), IV (vascular endothelium), and VIII (vascular endothelium). The post-translation modifications in terms of Vit-K dependent Υ carboxylation of glutamic acid residues allow them to bind with divalent cations particularly calcium. Most of these coagulation factors are enzyme precursors and exist as zymogen. The nomenclatures of these clotting factors were designated by Roman numeral and the activated form of these zymogens are designated by subscript “a” to their Roman numeral recommended by the international committee for the nomen­clature of blood clotting factors established in 1954. Initially

Table 4.17 Coagulation proteins/clotting factors

factor I to IX were officially approved.

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It is interesting to note that factor IX (Christmas), factor X (Stuart and Prower), factor XII (Hageman), Fletcher (prekallikrein), and Fitzgerald (high-molecular-weight kininogen) were named in the memory of six patients namely Stephen Christmas, Miss Audrey Prower and Rufus Stuart, John Hageman, Fletcher family, and Allen Fitzgerald as these factors were identified in those patients were suffering from coagulopathies associated with the absence of respective factors.

4.4.1.1 VascularEndothelium

The entire cardiovascular system including the capillaries are lined by a single layer of flattened cells known as the endothelium which separates blood from surrounding tissues. The endothelium comprised single-layered squa­mous cells (1-6 ? 10¹3 ) called endothelial cells of less than 0.2 μm thick with a total surface area of 4000-7000 m². There are some storage granules called Weibel-Palade bodies (WPBs) situated in the inner lining of the blood vessels and heart responsible for releasing two important hemostatic factor von Willebrand factor and P-selectin. The general functions of vascular endothelium include, regulation of vascular tone, proliferation of smooth muscle, molecular exchange between blood and surrounding tissue and regulation of hemostasis. Both anticoagulant and procoagulant factors are released from vascular endothelium which provides a hemocompatible environment to keep the blood in fluid state as well as initiates coagulation mechanisms during vascular injury. The functions of anticoagulation and procoagulation factors released from vascular endothelium are listed in Table 4.18.

Table 4.18 Anticoagulant and procoagulant factors of vascular endothelium

B. Procoagulants

4.4.2 Mechanism of Hemostasis

The blood coagulation or hemostatic mechanism can be divided into two broad events namely primary hemostasis in which there is formation of weak platelet plug and second­ary hemostasis where the reinforce of primary hemostasis is occurred with fibrin.

4.4.2.1 Primary Hemostasis

The formation of weak platelet plug in primary hemostasis is occurred through the events like vasoconstriction, platelet adhesion, platelet activation, and platelet aggregation.

Vasoconstriction: The very first response to vascular injury is the vasospasm which leads to vasoconstriction. The mediator of this vasoconstriction is endothelin-1 (ET) produced primarily from damaged endothelium. ET acts over the target tissue at the vascular smooth muscle after binding with its receptor coupled with G-protein. Binding of ET with its receptor causes the activation of phospholipase C (PLC) which converts phosphatidyl ino­sitol bis-phosphate (PI2) into di-acyl glycerol (DAG) and inositol tri-phosphate (PI3). Both DAG and IP3 stimulate the release of calcium from sarcoplasmic reticulum which causes muscle contraction.

Platelet adhesion: In this process, the rolling platelets are attached with the subendothelial layer exposed after vascular damage. Following the endothelial damage vWf, collagen, P-selectins are exposed which act as the ligands for binding with the membrane glycoprotein (GP) receptor situated in the phospholipid bilayer of plate­let membrane. The initial bond between platelet and subendothelial tissue is formed after binding of GpIb-IX receptor of platelets with vWf. The glycoprotein receptors of the platelets (GP VI) also bind with collagen. The attachment of platelet to the damaged surface facilitates platelet activation through intracellular signaling cascade. Platelet activation: The activation of platelet is mediated through the activation of either G protein coupled receptor (GP Ib-IX-V, GP VI) or C-type lectin-like receptor 2 (CLEC-2). GP VI is the major signaling receptor for platelet activation upon binding with collagen whereas CLEC-2 activates in response to rhodocytin, snake venom. Thrombin can also stimulate platelet activation through protease-activated receptors. Activation of platelets increases platelet intracellular calcium and triggers the contraction of microfilaments and moving of microtubules inward to compress the granules and release of their contents.

Platelet aggregation: The formation of platelet aggregates in the result of platelet activation and release of granular content together with the alterations of platelet shape from discoid to multiple pseudopodal plug which increase the surface area of platelets. The release of ADP from platelet granules induces the expression of GpIIb/IIIa complex. This complex plays a dual role in platelet adhe­sion (binding with vWF) and aggregation (binding with fibrinogen which acts as bridge between platelet-platelet aggregations). TXA₂ produced from activated platelets intensifies platelet aggregation which ultimately develops platelet plug.

4.4.2.2 Secondary Hemostasis

Weak platelet plug generates in primary hemostasis can only resist hemorrhage temporarily and needs further strengthening by the formation of fibrin which is the ultimate aim of secondary hemostasis. The events of secondary hemo­stasis include activation of coagulation cascade, formation of thrombin, and conversion of fibrinogen to fibrin. The classi­cal concept of blood coagulation described secondary hemo­stasis in three pathways, the extrinsic pathway, intrinsic pathway, and common pathway. The intrinsic pathway was so named because all the components required for coagula­tion were available in the blood itself and in contrary extrinsic mechanism required tissue factors from extravascular tissues. The detailed mechanisms will be discussed in subsequent sections. Both extrinsic and intrinsic pathways converge at the activation of factor X through different enzymatic reactions. Activated factor X then converts prothrombin to thrombin via common pathway. Thrombin initiates fibrin formation which stabilizes the clot. The entire process of secondary hemostasis requires Ca²⁺. The pathways are dia- grammatically represented in Fig. 4.5.

4.4.2.2.1 Formation of Fibrin Clot

The formation of fibrin is the end result of coagulation cascade. The fibrin is generated from the enzymatic cleavage of fibrinogen.

Enzymatic cleavage: Fibrinogen molecule is subjected to enzymatic cleavage of N-terminal fibrinopeptides known as fibrinopeptide A and fibrinopeptide B, respectively, from both the Aα and Bβ polypeptides.

Knob-hole interaction and formation of protofibrils: The removal of fibrinopeptides exposes “knobs” on the E domain, which can interact with the “holes” on the D domains. E domain on one fibrin molecule combines with the D domains on four other fibrin molecules resulting in the formation of staggered oligomers known as protofibrils.

Formation of fibrin clot: The protofibrils continue to aggre­gate and lengthen to form long fiber which then wind around to form multi-stranded bundles and ultimately gives rise to 3D network of fibrin clot.

Stabilization of fibrin clot: Thrombin activates FXIII which cross-links one fibrin molecule to another forming a strong bond to strengthen fibrin clot and protects it from physico­chemical damage.

4.4.3 Recent Concept of Blood CoagulationZCell-Based Model of Coagulation

The traditional model of blood coagulation stated the intrinsic and extrinsic pathway as separate cascade for generation of FX_a. But recent experiments suggests that extrinsic and intrinsic mechanism of blood coagulation can only explain

Fig. 4.5 Pathwaysofblood coagulation: Both intrinsic and extrinsic pathways facilitate the formation of prothombinase complex that converts prothrombin to thrombin. Thrombin converts fibrinogen to fibrin

Fig. 4.6 Cell-based model of blood coagulation: In the initiation phase, small amount of thrombin (IIa) is generated on the surface of TF bearing cells. The activation of factors Va, XIa, and VIIIa are occurred. In the propagation phase, large numbers of thrombin (IIa) are produced on the platelet surface

the coagulation mechanism for in vitro laboratory evaluation, but unable to explain the coagulation mechanism in vivo. The recent investigations on blood coagulation mechanism supports that the TF released from damaged endothelium is sufficient to initiate blood coagulation and the intrinsic mech­anism of coagulation does not have significant physiological role in hemostasis.

The cell-based model of coagulation describes the coagu­lation mechanism in the following ways (Fig. 4.6). injury followed by the formation of tenase and prothrombinase complex. The formation of tenase com­plex and prothrombinase complex allow the formation of large amount of thrombin catalyzes the formation of fibrin monomers. The monomers of fibrin then stabilize to form fibrin polymer and stable clot.

Initiation phase: This phase of coagulation occurs in the TF bearing cells outside the vasculature. In this phase, small amount of FIX and FX are activated by TF-FVII_a complex. Factor X_a converts prothrombin to thrombin associated with its cofactor FV_a. But it plays a pivotal role to initiate coagulation; firstly, it activates platelet to release its granule contents for platelet aggregation; secondly, it activates FV and FVIII on the surface of platelet which allows dissocia­tion of FVIIIa-vWF complex and make vWF free for platelet adhesion and aggregation at the site of injury; thirdly, it activates FXI on the surface of the platelets. It is believed that initiation phage of coagulation is always active in circulation and little amount of thrombin is con­tinually produced independent of vascular injury.

Elongation phase: After the vascular injury, platelets leave the blood vessel and interact with the exposed collagen at the extracellular matrix of injured site to form platelet plug.

Propagation phase: The propagation of clotting mechanism allows more and more platelets to adhere at the site of

4.4.3 Clot Retraction and Fibrinolysis

Clot retraction: Clot retraction denotes squeezing of clot through the contractile force generated by platelets results the expulsion of serum. The contraction of platelets is the result of interaction between platelet contractile proteins such as actin, myosin, and thrombosthenin. The contrac­tion of platelets further propagates through fibrin fibers to facilitate the reduction in clot volume. Clot retraction is essential to restore the blood flow at the site of injury vis-a-vis it promotes wound healing.

Fibrinolysis: The clot formation and fibrinolytic mechanism are well regulated under physiological condition to ensure the fluidity of blood during circulation. The fibrinolytic machinery consists of the activators, inhibitors, substrate and cofactors and the complex interaction amongst them facilitates fibrin degradation. The components of fibrino­lytic system and their mode of actions are depicted in Table 4.19.

Table 4.19 The components of fibrinolytic system

Initially plasmin acts over D-domain of the cross-linked fibrin polymer to release A«- and Bβ-fragments from C terminal region of « and β chain. The N terminal end of β chain is further cleaved to release fibrinopeptide B (FPB). A«- and B β-fragments together with FPB forms fragment X. Plasmin further cleaves three polypeptide bonds that con­nect D and E domain of fragment X to yield fragment D and fragment Y. The polypeptide bonds between D and E domain of fragment Y are cleaved to release individual D and E domain. The fragments thus generated by the action of plas­min upon the fibrin clot are collectively called fibrin degra­dation products (FDP). The principal component of FDP is D-dimer, the cross-linked product of two D fragments.

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