Mechanism Action of Platelets and Crucial Blood Coagulation Pathways in Hemostasis.

1When platelets decrease in number or become malfunction, the risk of hemorrhage is very high. Platelets, which circulate within the blood, are the essential mediators that trigger the mechanical pathway of the coagulation cascade upon encountering any damage to the blood vessels. Platelets encourage primary hemostasis via three major processes: activation, adhesion and aggregation. When the integrity of the vascular endothelium is interrupted, various macromolecular elements of the vascular subendothelium become exposed and readily accessible to platelets (Table 1)-.

2Hemostasis

3Hemostasis is a process to prevent hemorrhage by arresting and keeping the blood within the damaged vessel walls. Hemostasis is a complex process that is contingent on the complex interaction of platelets, plasma coagulation cascades, fibrinolytic proteins, blood vasculatures and cytokine mediators. Upon tissue injury, the hemostatic mechanism employs a plethora of vascular and extravascular receptors, in accordance with the blood components, to seal off the impairments to the vasculature and closing it off from the encircling tissues. Normal hemostatic responses can be organized into six different important phases, which fall under three major categories of hemostasis- (Table 2).

4Vasoconstriction

5Vascular spasm occurs whenever there is an injury or damage to the blood vessels. This will trigger a vasoconstriction, which could eventually stop the blood flow. This reaction can be responded within 30 minutes, and is localized to the injured area. At this stage, exposed collagen fibers will release ATP and other inflammatory mediators to recruit macrophages. In addition, the ECM becomes highly thrombogenicity, promoting platelet adhesion and aggregation-.

6Platelet plug formation

7Following vasoconstriction, exposed collagen from the damaged surface will encourage platelets to adhere, activate and aggregate to form a platelet plug, sealing off the injured area.

8Platelet adhesion

9The platelet adhesion mechanism is generally supported by the particular interactions between the membrane receptors and absorbed plasma proteins. The platelet membrane receptors are enriched with Gp receptors embedded in the phospholipid bilayer, including tyrosine kinase receptors, integrins, leucine rich receptors; G- protein coupled transmembrane receptors, selectins and immunoglobulin domain receptors. These are the important proteins involved to facilitate hemostatic function by mediating the interactions within cell-platelet and platelet-substrates-. The initial event that occurs upon hemostasis is the rolling and adherence of the platelets to the exposed subendothelium. Platelet adhesion is mediated by von Willebrand Factor (vWF) that binds to Gp Ib-IX in the platelet membrane. vWF is a blood Gp that serves as an adhesive protein, which could bind to other proteins, especially factor VIII at the wound sites, -,.

10Platelet activation

11A variety of stimuli can activate platelets. Platelet cells can also be activated upon biomaterial surface stimulation. Adhered platelets undergo degranulation and release cytoplasmic granules that contain serotonin, platelet activating factors and ADP. ADP is an important physiological agonist stored in the dense bodies of platelets that play an essential function in normal hemostasis and thrombosis. Platelets are activated to change shapes into a pseudopodal form upon the adhesion to the injured area which will activate the collagen receptors on their surface membrane, named GpIIbIIIa, to undergo release reactions. The GpIIbIIIa complex, organized through calcium-dependent association of GpIIb and GpIIIa that is a necessary step in platelet aggregation and endothelial adherence-. At the same time, platelets tend to synthesize and discharge thromboxane A2 (TXA2), aiding in vasoconstriction and platelet aggregation. In addition, GpIIbIIIa integrins and P-selectin move from the α-granule membrane to the platelet membrane to support platelet aggregation. Additionally, these are the receptors that could act as the catalytic surface and facilitate the hemostasis process-.

12Platelet aggregation

13Platelet aggregation begins once platelets become activated, triggering the GpIIbIIIa receptor (50-100/platelets), which attach to vWF or Fib. Each activated platelet extends pseudopods, clumping and becoming aggregated. These activations are further heightened by the generation of thrombin via the hemostasis mechanism. Platelet aggregation promotes a primary platelet plug. The ADP receptor interconnects with a family of ADP receptors (P2Y1 and P2Y12), which could be detected on platelets as helping with aggregation. P2Y1 receptors assist in stimulating the initial platelet shape changes and platelet aggregation. At the same time, P2Y12 is an important mediator for blood clotting. It increases significantly, responding to ADP to complete the aggregation process. Eventually, the formed platelet plug ought to be stabilized by the formation of fibrin, -.

14The coagulation mechanism

15Approximately fifty significant substances affect the blood coagulation mechanisms. The blood coagulation cascade of secondary hemostasis mainly consists of two main pathways: (i) intrinsic (contact activation pathway) (ii) extrinsic (TF pathway). The blood clotting process can be classified into three important steady steps as follows: (i) involvement of a complex cascade, triggering the chemical reactions that are mediated by the coagulation factors that respond to form fibrin strands for consolidating the platelet plugs; (ii) the conversion of prothrombin (PT) into thrombin, which is catalyzed by the PT activator; and (iii) conversion of Fib into fibrin, which eventually enmeshes the plasma, platelets and blood cells to build a firmer clot (Figure 6; Table 3)-.

16Extrinsic pathway

17The newer blood coagulation cascade model was well elaborated in a Jerry B. Lefkowitz. Thrombin was portrayed as the center of the coagulation universe. All the coagulation factors involved in the hemostasis process feed into the regulation and control of thrombin generation, which then forms clots at the sites of vascular injury. Thrombin is a proteolytic enzyme derived from PT, which aids in the process of forming blood clots by catalyzing the conversion of Fib to fibrin. The modified intrinsic coagulation cascade, which displayed in Figure 6, is different from the older one and lacks the significance of factor XII and prekallikren. Apparently, these proteins are not considered to play a crucial role in the coagulation process in vivo. There are two major processes that could initiate the blood clotting mechanism. They are extrinsic and intrinsic pathways.

18First, TF binds to factor VII or activated FVIII (FVIIIa) in 1:1 ratio complex. A limited proteolysis process extends to the TF / FVIIIa complex, which activates factor X or factor IX, further activating factor X/ FIX and activating serine proteases via cleaving an activation peptide. Proteolysis is the hydrolysis process that involves the breakdown of proteins into smaller polypeptides. Once the extrinsic pathway is triggered, the activation of factor X/ IX in the TF/ FVIIa complex is instantly inhibited by the TF pathway inhibitor (TFPI), which is generated from endothelial cells. Freshly activated factor IXa subsequently adheres to its cofactor, factor VIIIa, upon the phospholipid surface to stimulate the tenase complex which results in the activation of factor X to factor Xa.

19Finally, the common pathway for thrombin activation is initiated via the activation of factor Xa. The activated factor Xa merges with the cofactor, activated factor V (FVa) and calcium on the phospholipid surfaces to construct the prothrombinase complex. This complex eventually helps to convert PT to thrombin by cleaving the PT, which is the activation peptide. Thrombin activation will be generated to a very minor extent by the extrinsic pathway, which is adequate and crucial to initiate the coagulation cascade which subsequently triggers and expands thrombin generation via the intrinsic pathway-,.

20Intrinsic pathway

21The activation of factor XI to factor XIa, and more thrombin generated via factor IXa and factor VIIIa leads to the activation of factor X, which is involved in the intrinsic pathway. Factors V and VIII, which are partially proteolyzed or activated, are known to be involved in and facilitate the hemostasis process. Subsequently, the activation of factors V and VIII by thrombin triggers more mechanical action of the coagulation pathway by enhancing the bioactivity of tenase and prothrombinase complexes.

22As described in Table 3, factor I (Fib) plays a crucial role in forming a fibrin clot to seal the injured area with fibrin meshes. Fib typically consists of 3 globular domains, which is the central E domain attached or flanked by two identical D domains. At this stage, thrombin sticks to fibrinopeptides A and B, which are derived from the Aα and Bβ chains, to build a fibrin monomer. These monomers gather into protofibrils in a half-distributed manner, which is stabilized by the non-covalent interactions among fibrin molecules. Eventually, the protofibrils are obliquely organized into dense fibrin networks to form a temporary fibrin clot that is not covalently crosslinked.

23Nevertheless, to form a stable blood clot, thrombin needs to activate factor XIII to the transglutaminase enzyme activated factor XIIIa. Factor XIIIa will stimulate the glutamic acid and lysine side chains, producing a stable clot. Factor XIIIa is the fibrin stabilizing factor of the blood coagulation system that crosslinks with fibrin. Furthermore, factor XIIIa also plays a significant role towards tissue repair and the angiogenesis process (Figure 6)-,,.

24Tertiary hemostasis

25Once the fibrin clot has been formed, the activated platelets will be well organized and take position to contract their intracellular actin or myosin cytoskeleton. The intracellular actin network will directly connect to the integrin GpIIbIIIa and Fib receptor internally. Subsequently, the external component of GpIIbIIIa will adhere to the fibrin network of the blood clot, making the clot compact and decreasing the clot volume slowly, which is called clot retraction. A plasminogen activator is a serine protease that converts plasminogen to plasmin to promote fibrinolysis by cutting and degrading the fibrin networks. Plasmin slashes off the fibrin meshes formed around the wounded area, resulting in the formation of other circulating fragments that are cleared by other proteases or by the kidney and liver. The clot resolution mechanism helps in clearing the injured and obstructed vessels, regenerating blood flow that is directed to the normal blood flow pathway. GpIIbIIIa disrupts the fibrin binding capacity with platelets and complete the clot resolution process-,-.

26Wound healing

27Wound healing is an innate revitalizing response in tissue injuries, and the interaction of the cellular mechanical pathway events results in resurfacing, reconstitution and refurbishment of cells on the injured surface area. The healing process can be explained in three different phases followed by hemostasis, including inflammation, proliferation and maturation. While platelet play its crucial role in clot formation as part of the hemostasis process, the inflammatory cells aid in the inflammatory phase, which is followed by the proliferative phase, consisting of epithelialization, fibroplasia and angiogenesis. In addition, wound healing is also regulated by cytokines and growth factors. Collagen tends to form tight cross-links with other collagen

28and protein molecules in the maturation phase as well as completes the wound healing mechanism-.

29Cytokine is a protein mediator that is released from various cells. They can adhere to the cell surface receptors to trigger cell reactions and play a significant role in the wound healing phase. There are many cytokines responsible for wound healing, but Transforming growth factor-beta 1 (TGF-β1) and platelet derived growth factor- AB (PDGF-AB) are the main cytokines acting on cell proliferation and maturation. TGF-β1 helps to stimulate fibroblast proliferation and the production of proteoglycans, collagen and fibrin. It is also noted that TGF-β1 assists in decreasing scar formation and preventing inhibition of healing by glucocorticoids. On top of that, TGF-β1 supports the expression of specific cytokines in T cells to promote cell proliferation, especially while the cells still undergo the immature phase-.

30Meanwhile, PDGF-AB plays a specific role in blood vessel formation, which is addressed as an angiogenesis process. The angiogenesis process involves the growth of blood vessels from the existing blood vessel tissue. This is followed by the proliferation phase. The subsequent maturation phase replaces and remodels the structure of collagen type 3 to type 1. This maturation phase could last for a year or more than a year, depending on the severity of the wound type (Figure 7), -,.

31Platelets are the smallest blood component, that capable to act as a fundamental role in thrombosis and hemostasis. Initial platelet adhesion, activation and aggregation upon tissue injury, stimulates coagulation factors and other mediators to achieve hemostasis. In addition, these coordinated events are the vital biological processes towards wound healing upon the tissue damage. The history on the investigation of platelet function began 100 years ago, and has recently become increasingly important for monitoring hemorrhage, antiplatelet activity, platelet dysfunctions and coagulation factors. Understanding the platelet thrombogenicity cascade is very beneficial to reducing the hematological complications. As platelets are remarkable cells that arrest bleeding and circulate within the human body, the search for artificial platelet substitutes should be part of the next attempt or search to decrease platelet-related morbidities and mortalities.