Process of formation of the blood clot

Platelets in Blood Clotting

Blood clot formation typically progresses through three phases: initiation, extension, and stabilization (Figure 2). When given the appropriate signals, usually by damaged blood vessel walls, platelets undergo activation and initiate formation of a blood clot. Platelets initiate blood clotting by tethering to and literally rolling on damaged endothelial cells at sides of blood vessel injury. This interaction is mediated by von Willebrand factor and collagen that are exposed on the damaged vessel wall binding to GPIb-IX-V on the platelet surface. If this binding is strong enough the platelets will sit down and spread onto the injured surface and form a monolayer of adhesive platelets (Brass, 2010).

Extension occurs when the first layer of platelets become activated and releases substances that attract and activate other platelets. The most potent of these are thrombin, ADP, and thromboxane-A2. ADP is secreted from the platelet dense granules, thromboxane-A2 is generated from the lipids of the activated platelet membrane, and thrombin is generated by the plasma coagulation proteins on the surface of the activated platelets. This second wave of platelets binds to the first monolayer and extends the clot outward from the vessel wall. This results in further release of procoagulant substances and activation of the platelet surface protein αIIbβ3. Upon activation, the αIIbβ3 surface receptor undergoes a conformational change into an active, highly adhesive form that can bind fibrinogen, von Willebrand factor, and other large proteins in the plasma. These proteins are large enough to reach from platelet to platelet, which leads to crosslinking and extension of the clot in three dimensions. Simultaneously, the thrombin activated by the coagulation factors generates a fibrin mesh that binds the thrombus together. Finally, factor XIII, which is also secreted by activated platelets, crosslinks the fibrin mesh, solidifying the clot.

Abstract

The coagulation cascade leads to clot formation to prevent blood loss. In physiological conditions, there is a balance between procoagulant and anticoagulant systems. This balance is disturbed in pathological conditions such as thrombosis or pathological bleeding that may be dangerous for patients. Anticoagulants are prescribed to treat both arterial and venous thromboembolic diseases, which are a common cause of morbidity and mortality worldwide, and antiplatelet drugs are used to treat and prevent arterial thromboembolism. In this chapter, we summarize the most important anticoagulants and antiplatelet drugs that are used in clinical practice and explain their pharmacodynamics including mechanism of actions, efficacy, and other possible mechanisms beyond antithrombotic effects; pharmacokinetics including absorption, distribution, metabolism, and excretion; therapeutic values in different conditions including elder patients, renal and hepatic impairments, and pregnancy; the measurement of antithrombotic activities, major interactions, and toxicities. Also, the future outlook of these agents is discussed.

The Process of Wound Healing

The immediate tissue response to wounding is clot formation to stop bleeding. Simultaneously, there is a release of inflammatory cytokines that regulate blood flow to the area, recruit lymphocytes and macrophages to fight infection, and later stimulate angiogenesis and collagen deposition (Williams and Kupper, 1996). These latter processes result in the formation of granulation tissue, a highly vascularized and cellular wound connective tissue. Fibroblasts rich in actin, called myofibroblasts (Desmouliere and Gabbiani, 1996), are recruited through the action of factors such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β). Granulation tissue forms in the wound bed, stimulated by factors such as PDGF. This tissue is gradually replaced by scar tissue through the action of the myofibroblasts and factors such as TGF-β. Keratinocytes are stimulated to proliferate and to migrate into the wound bed to restore epidermal coverage. From our preclinical observations using both full-thickness HSE and dermal matrices, coverage by the epidermis appears to play a key role in the regulation of the underlying inflammatory response. Providing a noninflammatory living connective tissue implant in the wound defect also appears to be beneficial in directing the granulation response.

Why should living tissue be different in its response? The epidermal and dermal response is regulated by inflammatory cytokines and by autocrine and paracrine factors produced by the dermal fibroblasts and epidermal keratinocytes (Ansel et al., 1990; McKay and Leigh, 1991). These factors regulate growth and differentiation of keratinocytes, proinflammatory reactions, angiogenesis, and deposition of extracellular matrix. Living tissue created through tissue engineering can provide complex temporal control of factor delivery and effect and can be used to provide the needed combination of chemical, structural, and, last but not least, normal cellular elements (Sabolinski et al., 1996).

As this brief description shows, wound healing involves the interaction of many tissue factors and elements. The poor healing response in chronic wounds has been attributed to an imbalance of factors rather than to an insufficiency of any particular factor (Parenteau et al., 1997). However, most, if not all, factor-based approaches have had marginal success. Identification of putative wound-healing factors has led to several attempts to speed wound healing by local application of one or more factors that promote cell attachment and migration. Transforming growth factor-β (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) have been candidates for this purpose (McKay and Leigh, 1991; Abraham and Klagsbrun, 1996). Of these, only PDGF has shown efficacy in clinical trials and is approved for clinical use (Regranex®, Ortho McNeil, Inc., Raritan, NJ 08869-0602). The arginine-glycine-aspartic acid (RGD) matrix peptide sequence has been found to promote the migration of connective tissue cells and, thus, to stimulate production of a dermal scaffold within the wound bed. This approach has been shown to accelerate healing of sickle-cell leg ulcer and diabetic ulcers (Steed et al., 1995), as compared with placebo but not when compared with standard care. In addition, complex cell extracts have been used in hopes of providing the appropriate mixture of elements. These include the use of platelet extract to provide primarily platelet-derived growth factor (PDGF) and the use of keratinocyte extracts to provide a complex mixture of elements of rapidly growing keratinocytes — again, with marginal effect, in part due to the complex nature of the wound healing response. In addition, the use of factors is not a sufficient approach, in and of itself, in situations where there is severe or massive loss of skin tissue.

Abstract

One example demonstrating the significance of studying cell mechanosensing is the platelet-mediated blood clot formation (thrombosis) under shear that causes heart attack and stroke—the No. 1 killer worldwide. As an emerging concept, shear forces generated by the hemodynamic flow disturbances have been shown to promote rapid platelet aggregation, which is usually seen in a narrowing vessel due to a growing thrombus, an atherosclerotic plaque, or an interventional device. Notably, this biomechanical mechanism not only challenges the classic agonist-dependent (biochemical) thrombosis model but also demonstrates resistance to the existing antithrombotic agents. Furthermore, recent studies indicate that this biomechanical platelet aggregation requires the interaction of platelet receptor glycoprotein Ibα (GPIbα) with von Willebrand factor (VWF) in plasma. This chapter will summarize the latest understandings on the platelet mechanosensing principles that center the VWF–GPIbα axis. Clinically, this platelet mechanosensing pathway represents a novel therapeutic target for an anti-thrombotic purpose.

Osteochondral injuries

Unlike injuries limited to cartilage, injuries that fracture subchondral bone cause hemorrhage and fibrin clot formation and activate the inflammatory response [43–45]. Soon after injury, blood escaping from the damaged-bone blood vessels forms a hematoma that temporarily fills the injury site. Fibrin forms within the hematoma and platelets bind to fibrillar collagen. A continuous fibrin clot fills the bone defect and extends for a variable distance into the cartilage defect. Platelets within the clot release vasoactive mediators and growth factors or cytokines (small proteins that influence multiple cell functions, including migration, proliferation, differentiation, and matrix synthesis). These cytokines include transforming growth factor beta and platelet-derived growth factor. Bone matrix also contains growth factors, including transforming growth factor beta, bone morphogenic protein, platelet-derived growth factor, insulin-like growth factor I, insulin-like growth factor II, and others. Release of these growth factors may have an important role in the repair of osteochondral defects. In particular, they stimulate vascular invasion and migration of undifferentiated cells into the clot and influence the proliferative and synthetic activities of the cells. Shortly after entering the tissue defect, the undifferentiated mesenchymal cells proliferate and can begin to synthesize a new matrix. Within 2 weeks of injury, some mesenchymal cells assume the rounded form of chondrocytes and begin to synthesize a matrix that contains type II collagen and a relatively high concentration of proteoglycans. These cells produce regions of hyaline-like cartilage in the chondral and bone portions of the defect. Six to eight weeks after injury, the repair tissue within the chondral region of osteochondral defects contains many chondrocyte-like cells in a matrix consisting of type II collagen, proteoglycans, some type I collagen, and noncollagenous proteins. Unlike the cells in the chondral portion of the defect, the cells in the bony portion of the defect produce immature bone, fibrous tissue, and hyaline-like cartilage. Over time, this tissue remodels form normal bone.

The chondral repair tissue typically has a composition and structure intermediate between that of hyaline cartilage and fibrocartilage, and it rarely, if ever, replicates the elaborate structure of normal articular cartilage [1,46–48]. Occasionally, the cartilage repair tissue persists unchanged or progressively remodels to form a functional joint surface. But in most large osteochondral injuries, the chondral repair tissue begins to show evidence of depletion of matrix proteoglycans, fragmentation and fibrillation, increasing collagen content, and loss of cells with the appearance of chondrocytes within a year or less. The remaining cells often assume the appearance of fibroblasts as the surrounding matrix comes to consist primarily of densely packed collagen fibrils. This fibrous tissue usually fragments and often disintegrates, leaving areas of exposed bone. The inferior mechanical properties of chondral repair tissue may be responsible for its frequent deterioration [1,44]. Even repair tissue that successfully fills osteochondral defects is less stiff and more permeable than normal articular cartilage, and the orientation and organization of the collagen fibrils in even the most hyaline-like cartilage repair tissue do not follow the pattern seen in normal articular cartilage. In addition, the repair tissue cells may fail to establish the normal relationships between matrix macromolecules, in particular, the relationship between cartilage proteoglycans and the collagen fibril network. The decreased stiffness and increased permeability of repair cartilage matrix may increase loading of the macromolecular framework during joint use, resulting in progressive structural damage to the matrix collagen and proteoglycans, thereby exposing the repair chondrocytes to excessive loads and further compromising their ability to restore the matrix.

Clinical experience and experimental studies suggest that the success of chondral repair in osteochondral injuries may depend to some extent on the severity of the injury, as measured by the volume of tissue or surface area of cartilage injured and the age of the individual [49]. Smaller osteochondral defects that do not alter joint function heal more predictably than larger defects that may change the loading of the articular surface. Potential age-related differences in healing of chondral and osteochondral injuries have not been thoroughly investigated, but bone heals more rapidly in children than in adults, and the articular cartilage chondrocytes in skeletally immature animals show a better proliferative response to injury and synthesize larger proteoglycan molecules than those from mature animals [40,50–54]. Furthermore, a growing synovial joint has the potential to remodel the articular surface to decrease the mechanical abnormalities created by a chondral or osteochondral defect.

3.5 Rotational thromboelastometry

Rotational thromboelastometry (ROTEM) provides a point-of-care analysis of the viscoelastic properties of clot formation and dissolution. It is assumed to be superior to screening coagulation tests, such as PT and APTT, in acute traumatic coagulopathy, because it includes the contribution of blood cells and provides information about the clot stability [44,45]. In the ROTEM assay, coagulation can be triggered in different ways, but most commonly with a contact activator and calcium (the INTEM test), similar to APTT, or with thromboplastin (the EXTEM test), similar to PT. Whole citrated blood is pipetted into a cup in which a rotating pin is inserted. An optical system detects the impedance of the rotation of the pin and plots the trace produced by the viscoelastic changes associated with fibrin clot formation. From this tracing several parameters can be recorded: clotting time (CT), clot formation time (CFT), speed of clot formation (the alpha angle), amplitude 10 min after CT (A10), maximum clot firmness (MCF), lysis index 30 min after CT (LI30) and maximum lysis (ML). There is not much known about the effect of different anticoagulant drugs on different ROTEM tests. Most of the published studies were in vitro studies utilizing blood or plasma samples spiked with anticoagulants (e.g., Refs. [46,47]). Although valuable, data from these studies cannot be extrapolated to patients receiving anticoagulant drugs [48].

Unfractionated heparin prolongs the INTEM CT and the presence of heparin can be confirmed with the addition of heparinase (HEPTEM), which shortens the CT. The effect of LMWH on ROTEM has been tested in an in vitro study in which two LMWHs both prolonged the INTEM CT significantly, but with great variability [48]. VKAs prolong the EXTEM CT, but only around and above the upper therapeutic range level [49,50]. Little is known about the effect of direct oral anticoagulants. One study reported dose-dependent relationship between dabigatran concentration and INTEM, EXTEM, FIBTEM and APTEM CT in patients with atrial fibrillation receiving either 110 or 150 mg dabigatran twice daily. A statistically significant correlation between dabigatran and MCF has also been reported, but not yet explained [51].

2.2.1 Heparin

The extensive amount of thrombin formed during heart surgery needs to be antagonized in order to avoid intravascular clot formation as well as coagulation of the CPB circuit (namely localized in the sites of blood stagnation such as the venous reservoir filtering nets). Since the very beginning of cardiac surgery, heparin has been used to avoid the conversion of fibrinogen to fibrin triggered by thrombin. Unfractionated heparin is a cofactor accelerating the reaction between thrombin (and factors IXa, Xa, XIa, Xlla) and its natural antagonist antithrombin (AT). Heparin speeds up thrombin inhibition, but fails to prevent thrombin formation3, 4 and cannot inhibit clot-bound thrombin.19 Unfractionated heparin is rapidly acting and rapidly reversed by protamine; however, this drug has many disadvantages, the most important of which are related to it being very difficult to predict the patient’s heparin responsiveness. There is a wide variability of heparin efficacy in terms of thrombin inhibition, resulting from (a) different amounts of active heparin molecules in different commercial preparations, depending on the presence of the AT-binding pentasaccharide,20 (b) the AT activity,21, 22 (c) the platelet count, there being lower efficacy in patients with thrombocytosis,22 other patient-related conditions, all decreasing heparin responsiveness, such as advanced age and diabetes.23

Conventionally, the loading dose of heparin for establishing CPB ranges between 300 and 400 IU kg–1. Heparin concentration is difficult to monitor during the operation,2 and its anticoagulant action is usually assessed with the activated clotting time (ACT).

9.2.4 Fibrinogen

The Clauss assay is the most widely employed method for the measurement of functional fibrinogen, measuring fibrinogen-dependent clot formation.17 With this assay, fibrinogen concentration is inversely proportional to the time recorded for clot formation. The Clauss assay is commonly performed on an automated coagulometer, although a simple, single-use microfluidic device was reported by Dudek et al.18 who claim to measure fibrinogen concentration in the normal range (1–7 g/L) with a turnaround time of 5 min. The concentration of fibrinogen is related to the distance travelled by the sample on a lateral flow device that contains biological activators of clotting.18

Fibrinogen measurements are also recorded in the central laboratory using the prothrombin time-fibrinogen (PT-Fg), which is an assay based on clotting time. Fibrinogen concentrations are measured because of the change in optical density or light scatter on an automated coagulometer.19 Although few POC tests have been developed in this space for human diagnostics, POC fibrinogen assays are popular in veterinary diagnostics. One example is the Solo Fibrinogen Test (Euro Lyser Diagnostica) which uses an immunoturbidimetric assay and light scatter, all incorporated into a miniaturised bench-top instrument and returning a result within 8 min.

Acute versus chronic wound healing

One of the basic differences between acute and chronic wounds is that in the former the sequence of steps and phases involved (clot formation, inflammation, migration and proliferation, and remodeling) occur in a very orderly and linear fashion. Such is not the case in chronic wounds, where there is a fundamental asynchrony of the healing process. Within the chronic wound, the various phases of wound repair may be occurring at the same time, or not in the appropriate sequence. Wound bed preparation is a way to get the chronic wound to behave more like an acute wound. Often, surgical debridement is all that is required. At other times, treatment of bacterial infection, removal of edema, etc., are essential additional steps [11].

Acute versus chronic wound healing

One of the basic differences between acute and chronic wounds is that in the former the sequence of steps and phases involved (clot formation, inflammation, migration and proliferation, and remodeling) occurs in a very orderly and linear fashion. On must be fully aware of the fact that the often cited steps involved in wound healing come from observations in experimental animal wounds, generally in mice and rats. Therefore, at least theoretically, the steps may be significantly different in human wounds. Anyhow, the orderly steps involved in acute wounds are not the same as in chronic human wounds, where there is a fundamental asynchrony of the healing process. Within the chronic wound, the various phases of wound repair may be occurring at the same time, or not in the appropriate sequence. WBP is a way to get the chronic wound to behave more like an acute wound. Often, surgical debridement is all that is required. At other times, treatment of bacterial infection, removal of edema, etc., are essential additional steps [11].