Fibrin Formation

Anticoagulant and Antiplatelet Therapy

David Royston , in Pharmacology and Physiology for Anesthesia, 2013

Mechanisms of Thrombin and Fibrin Generation

The coagulation phase of hemostasis involves generation by thrombin of fibrin, which binds and stabilizes the weak platelet hemostatic plug. Thrombin is highly specific in cleaving fibrinogen at only two arginine sites. The active site of thrombin is surrounded by negatively charged amino acid residues and away from this are positively charged exosites. This arrangement allows the very specific alignment and cleavage of fibrinogen (Figure 37-1).

There are no covalent bonds holding platelets together during the formation of the primary hemostatic plug. If left in this state, the platelet plug disintegrates in a few hours, resulting in late bleeding. The process of blood coagulation, with soluble factors in the blood entering into a cascade of protease activation that leads to the formation of fibrin, is localized to the site where the original platelet plug was formed. This localization is achieved by two methods. First, the chain of reactions that leads to conversion of fibrinogen to fibrin is restricted to a surface, such as platelet phospholipids. Second, a series of inhibitors constrains the reaction to the site of injury and platelet deposition.

Historically the blood coagulation system is divided into two initiating pathways: the tissue factor (extrinsic) pathway and the contact factor (intrinsic) pathway. These pathways meet in a final common pathway whereby factor Xa converts prothrombin to thrombin, which then cleaves fibrinogen to fibrin. The prothrombin time (PT) is a plasma and test tube test of the integrity of the extrinsic pathway, and the activated clotting time (ACT) or activated partial thromboplastin time (aPTT) are tests of the intrinsic system for blood and plasma, respectively.

This model based on the concept of a waterfall or cascade is an over-simplification of the coagulation system, as proteins from each pathway influence one another. It is probably more correct to think of the coagulation system as an interactive network with carefully placed amplifiers and restraints.

Fibrin formation is a process of initiation and amplification. The specific properties of platelets and the coagulation system cooperate to ensure that fibrin formation occurs only at the localized site where it is required to initiate wound repair. This is achieved by a number of physicochemical means:

1

The surface of resting platelets contains acidic phospholipids such as phosphatidylserine that have their negatively charged pole directed inward. During irreversible shape change, this pole is flipped to the outside of the platelet to provide a negatively charged outer surface.

2

The coagulation system relies primarily on soluble factors synthesized in the liver that circulate in the plasma in an inactive, zymogen form and become active after proteolytic cleavage. Apart from factor XIII, which is a transglutaminase, all active factors are serine proteases related to the digestive enzyme trypsin. Other factors in the coagulation process, such as tissue factor, factor V, factor VIII, and high molecular weight kininogen (HK) act as cofactors.

Factors VII, IX, X, and prothrombin contain carboxylated glutamic acid residues at their N-terminal regions. Vitamin K acts as a cofactor for the enzyme that carboxylates glutamic acid, forming gamma-carboxy glutamic acid, with a resultant higher density of negative charges. This charged area interacts at the organizing surface of the platelet with ionized Ca2+, acting as a bridge with the negative surface charge on the activated platelet.

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Hormone Therapy and Hemostasis

MORRIS NOTELOVITZ , in Treatment of the Postmenopausal Woman (Third Edition), 2007

V FIBRIN FORMATION AND FIBRINOLYSIS

The introduction of t-PA therapy into clinical practice emphasizes the importance of fibrinolysis in maintaining intimal health and vascular patency. Fibrinogen is a dimer with three polypeptide chains: α, β, and γ. Thrombin acts on the a and β chains and produces two molecules of fibrinopeptide A and two molecules of fibrinopeptide B. This leaves a large residue molecule known as fibrin monomer, which spontaneously polymerizes into non-stable fibrin polymer. These are cross-linked in the presence of calcium and activated factor XIII to form the stable insoluble clot, fibrin (9). This process is vital to survival and is nature's way of preserving the integrity of the vascular tree subsequent to injury so that healing and normal function of the area concerned can be restored and maintained.

Fibrin formation is regulated by the process of fibrinolysis, which involves the enzymatic degradation of fibrin and fibrinogen by plasmin ( 9). Plasmin is formed from plasminogen, a β-globulin synthesized by the liver (25). The biologic activity of plasmin, and hence fibrinolysis, is determined by activators and inactivators of plasminogen and plasmin inhibitors (see Fig. 35.1). Fibrinolysis is initiated by either factor XIIa or urokinase plasminogen activator (intrinsic pathway) or tissue-type plasminogen activator (t-PA), the extrinsic pathway (31). Tissue plasminogen activator, which is produced by the endothelial cells and released into the circulation, in the presence of fibrin binds together with plasminogen to generate plasmin, and thus fibrinolysis (17). Fibrin acts both as a substrate and a cofactor to plasminogen activity.

FIGURE 35.5. Diagrammatic depiction of how fibrin polymer forms. Asterisks depict potential markers for thrombosis.

Tissue plasminogen activator is inhibited by plasminogen activator inhibitor type 1 (PAI-1) (32). PAI-1 is produced by hepatocytes and endothelial cells. The balance between t-PA and PAI-1 is said to be the major determinant of the spontaneous fibrinolytic activity of blood (32). Both fibrinogen and PAI-1 are significantly reduced by combination transdermal estradiol plus MPA, and the mean plasma estradiol level was found to be 138 pg/mL (33). In another study, conjugated estrogen (alone and in combination with MPA) significantly reduced plasma PAI-1 antigen levels (34). In this study, transdermal estrogen had very little effect on PAI-1.

As noted, plasminogen has to bind to fibrin in order to be activated to plasmin. The binding takes place at the so-called "lysine-binding" sites on the plasminogen molecule. Histidine-rich glycoprotein has an affinity for these sites and can, therefore, control the amount of biologically available free plasminogen (35). Estrogen decreases plasma histidine-rich glycoprotein and may, therefore, enhance fibrinolysis (36). Exogenous progestins and estrogen (37) are associated with increased plasminogen activity (5). Three main plasmin inhibitors exist: α2-plasmin inhibitor; α2-macroglobulin, which reacts quickly and as a competitive plasmin inhibitor; and α2-antitrypsin, which reacts more slowly but more firmly. The latter two proteases also inhibit thrombin and as such have conflicting functions: they prevent clot formation by antagonizing thrombin, and they encourage fibrin and fibrinogen integrity by inhibiting plasmin. Their overall effect on thrombogenesis is not known. Antiplasmin inhibitor (α2-plasmin inhibitor) is said to inhibit 35% of the plasmin generated from plasminogen (38). It acts in two ways: direct inactivation of plasmin and blockage of plasminogen binding to fibrin. Pharmacologic lowering of α2-plasmin inhibitor levels can be viewed as a positive side effect because the net effect will result in increased fibrinolysis.

Cleavage of fibrin and fibrinogen produces a variety of fragments known as fibrin or fibrinogen degradation products (FDPs). FDPs have potent anticoagulant properties and may interfere with platelet activity as well.

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Acquired Coagulopathy

K. Tanaka , D. Bolliger , in Reference Module in Biomedical Sciences, 2014

Restoration of Clotting Factors

Fibrin formation and thrombin generation are two pivotal processes in hemostasis, and therefore it is clinically important to restore fibrinogen and prothrombin levels. The thresholds for fibrinogen replacement have been conventionally set at 80–100 mg dl −1 based on the clinical data from congenital afibrinogenemia. However, higher cut-off values (150–200 mg dl−1) have been advocated in Europe based on the clinical experience with purified human fibrinogen concentrate in perioperative coagulopathy. The body size and plasma fibrinogen levels prior to the vascular injury can be important determinants of susceptibility to dilutional coagulopathy. Two liters of blood loss and subsequent fluid replacements are more likely to result in clinically significant hemodilution in a 50 kg female compared to a 90 kg male. A dramatic increase of plasma fibrinogen during pregnancy may be regarded as a natural protection against postpartum hemorrhage. Indeed, those women with prepartum fibrinogen levels ≤200 mg dl−1 are more likely to have postpartum bleeding complications. Dilutional coagulopathy tends to occur early and becomes severe in neonates and infants during major trauma and invasive surgical procedures (e.g., extracorporeal circulation).

Prothrombin levels are a key determinant of thrombin generation, which is crucial for localized platelet activation, fibrin polymerization, and clot stabilization. Although the minimal prothrombin level is considered to be around 20% in isolated (congenital) prothrombin deficiency, 50–60% (of normal activity) may be required for adequate hemostasis in the presence of multifactorial deficiency and thrombocytopenia. In addition, FXIII functions as an important clotting enzyme at the end of coagulation cascade. Thrombin-activated FXIII becomes a transglutaminase, which cross-links α2-antiplasmin to fibrin, and polymerizes fibrin monomers. The minimal FXIII levels of 50–60% are suggested from the recent clinical data in case of major perioperative bleeding.

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Carotid Endarterectomy

M.J.G. Harrison , in Topical Reviews in Neurosurgery, 1982

Anticoagulants

The prevention of fibrin formation by anticoagulation has proved to be hazardous and of little value in the aftermath of completed strokes due to infarction. In the case of TIAs, several trials were carried out but were either small or not randomized. Attempts to combine the figures from the few randomized studies, or to use natural history studies as controls, suggest a modest benefit in reduction of TIAs [ 84] and less certainly a reduction in stroke [85, 86]. In none of the randomized trials was the reduction of strokes by anticoagulants statistically significant. There is some evidence that the risk of stroke in the first few months after a TIA is reduced [87], whilst haemorrhagic complications are unusual before 12 months. Some physicians who still employ anticoagulants have therefore changed to a short term use. There is, however, an increased risk of TIA and stroke on withdrawal of treatment [86].

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Advances in monitoring anticoagulant therapy

Mojca Božič Mijovski , in Advances in Clinical Chemistry, 2019

2.3 Thrombin time

Thrombin time (TT) measures fibrin formation caused by the action of thrombin—the last step in the coagulation cascade. The principle of the test is that a standardized concentration of thrombin is added to citrated plasma and time to fibrin clot formation recorded in seconds. Reference range depends on the thrombin concentration used.

Unfractionated heparin causes a considerable prolongation of TT, but is not routinely used for therapy monitoring. It may be used for qualitative assessment of the unfractionated heparin presence in blood or when blood sample contamination with heparin is suspected, i.e., when blood is drawn from a central line. LMWH, fondaparinux or direct factor Xa inhibitors have no effect on TT as they predominantly or exclusively inhibit factor Xa. VKAs, too, have no effect on TT.

Direct thrombin inhibitors, such as bivalirudin, argatroban and dabigatran, prolong TT considerably. This prolongation becomes unmeasurable by most automated coagulation analyzers already at low thrombin inhibitor concentrations: in the case of dabigatran, at concentrations between 40 and 100   ng/mL. TT cannot be used for monitoring therapy with direct thrombin inhibitors, but TT within the reference range reliably excludes clinically relevant direct thrombin inhibitor concentrations in blood.

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ATHEROSCLEROSIS AND THROMBOSIS IN DIABETES MELLITUS: NEW ASPECTS OF PATHOGENESIS

JOHN A. COLWELL , ... RUDOLF J. JOKL , in Levin and O'Neal's The Diabetic Foot (Seventh Edition), 2008

Coagulation

Activation of the coagulation system leads to fibrin formation by thrombin. Experimental and clinical data suggest that primary fibrin deposits and mural thrombi lead to the initial endothelial lesion and may contribute to the development of macrovascular and microvascular disease. In diabetes, there may be a general activation of blood coagulation, and fibrin lesions can be found in several organs of diabetic subjects. Most of the individual factors of both the intrinsic and the extrinsic coagulation pathway, as well as the inhibitors of coagulation, may be altered in diabetes. There are multiple data to support a pathogenetic rather than consequential role of hypercoagulation in the development of vascular disease in diabetes.

Attention has been directed at fibrinogen levels and dynamics in diabetes for a variety of reasons. It is now clear that the plasma level of fibrinogen is an independent risk factor for thrombotic and major cardiovascular events in population-based studies. 277 There have been many studies of fibrinogen levels and dynamics in diabetes mellitus. Generally, plasma fibrinogen levels are found to be elevated in diabetic individuals, particularly in those with previous hyperglycemia. Insulin deficiency results in an increase in fibrinogen synthesis in type 1 diabetes, and an insulin infusion will decrease the fibrinogen synthetic rate. Fibrinogen survival has been reported to be decreased in diabetes, and this abnormality is quickly reversed when euglycemia is achieved with insulin. Decreased fibrinogen survival in diabetes is also reversed by heparin, suggesting that intravascular fibrin formation may be taking place. Fibrinogen is glycated in diabetic subjects, and cross-linking of the α-chains of fibrinogen is impaired. Exercise conditioning will lower plasma fibrinogen levels in type 2 diabetic individuals. In the DCCT/EDIC cohort, fibrinogen was identified as a marker of nephropathy and peripheral vascular disease in type 1 diabetics. 278 These findings suggest that there might be increased fibrin formation in vivo in individuals with diabetes.

Because fibrinogen to fibrin formation is catalyzed by thrombin, investigations have centered on the regulation of thrombin activity in diabetes and on an in vivo index of thrombin activity, fibrinopeptide A (FPA). 279 FPA is cleaved from the α-chain of fibrinogen by the action of thrombin. This forms the first step in the conversion of fibrinogen to fibrin. FPA levels tend to be elevated in diabetes, especially when control is poor or vascular problems exist. Furthermore, recent studies have indicated that elevated FPA levels might be seen in diabetic individuals before vascular complications are present. A relation between plasma and urinary FPA and hyperglycemia in diabetes has been reported.

Prothrombin activation fragment F1+2 has been identified as a sensitive marker of coagulation in vivo. F1+2 is released from prothrombin when it is converted to thrombin by activated factor X. (Intima-media thickness, an index of subclinical atherosclerosis, is strongly associated with plasma F1+2 in the general population. 280 ) F1+2 generation proved to be very sensitive to even a short increase of glycemia. 281 Positive correlation between F1+2 and glycated hemoglobin in diabetic patients has been found. 282

The most important inhibitor of the coagulation system is antithrombin III (AT III). AT III activity may be modulated by glucose both in vitro and in vivo. Hyperglycemia will cause a decrease in AT III activity in nondiabetic subjects, and activity returns to normal after a glucose infusion is stopped. Depressed levels of AT III activity are found in adult type 1 diabetic subjects, and infusion of insulin to produce normoglycemia will return AT III activity to normal. Nonenzymatic glycosylation of the AT III molecule might be the cause of its impaired functional activity. 283

Activated protein C is a vitamin K–dependent plasma protein that is another potent anticoagulant. It acts at the level of factors V and VIII in the intrinsic coagulation scheme. Several investigators have reported decreased protein C antigen and activity levels in type 1 diabetes, and such changes could theoretically promote coagulation. Glucose-induced hyperglycemia will lead to a fall in protein C levels and activity in normal and diabetic individuals. Depressed plasma levels and activity of protein C will rise with insulin-induced normoglycemia in type 1 diabetes.

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Platelets, Coagulation, and the Liver

Louise C. Kenny , ... F.Gary Cunningham , in Chesley's Hypertensive Disorders in Pregnancy (Fourth Edition), 2015

Fibrinolytic System

The end-product of the coagulation cascade is fibrin formation. The main function of the fibrinolytic system is to remove excess fibrin deposited as a consequence of thrombin activity (see Fig. 17.1). Plasmin, which is the main protease enzyme in this system, originates from plasminogen secreted by the liver. The activation of plasminogen into plasmin is through plasminogen activators which are serine proteases. These include tissue- and urokinase-plasminogen activators – t-PA and u-PA – and they are secreted by endothelial cells, monocytes, and macrophages. Both act upon plasminogen to convert it to plasmin and in so doing trigger a proteolysis cascade that causes thrombolysis. Plasmin cleaves and converts t-PA and u-PA into two-chain proteases, which exhibit higher proteolytic activity, implying a positive feedback for the fibrinolytic cascade. 141

A negative feedback is therefore essential for the fibrinolytic pathway and thus the activity of plasminogen activators is balanced by plasminogen activator inhibitors. Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor that functions as the principal inhibitor of t-PA and u-PA. The other PAI, plasminogen activator inhibitor-2 (PAI-2), is secreted by the placenta and is only present in significant amounts during pregnancy. In addition, plasminogen activator inhibitor-3 (the protease nexin) acts as an inhibitor of t-PA and urokinase.

There are other fibrinolytic inhibitors. A2-antiplasmin (α2-AP) is a single-chain glycoprotein that reacts with plasmin to form a plasmin–α2-AP complex which is incapable of breaking down fibrin. Thrombin activatable fibrinolytic inhibitor – TAFI – is a glycoprotein synthesized by the liver and also found in platelet granules and it acts as an enzyme that may modulate fibrinolytic activity. Finally, α2-macroglobulin – α2-M – is synthesized mainly by the liver and is a general inhibitor of both coagulation and fibrinolysis, acting as a scavenger. In the fibrinolytic system, α2-M inhibits the action of plasmin and kallikrein, while in coagulation it inhibits thrombin.

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Prevention and Treatment of Venous Thromboembolism

Michael B. Streiff MD , in Consultative Hemostasis and Thrombosis (Fourth Edition), 2019

Parenteral Direct Thrombin Inhibitors

Thrombin is a critical serine protease in the coagulation cascade that catalyzes fibrin formation from fibrinogen and activates factors V, VIII, and XIII, as well as platelets. Because traditional anticoagulants such as UFH are indirect thrombin inhibitors, there has been considerable investigation into developing direct thrombin inhibitors that do not require antithrombin III for activity. Lepirudin, the first widely available parenteral direct thrombin inhibitor, was a recombinant form of hirudin that was US Food and Drug Administration (FDA) approved for treatment of HIT. 111,112 It has since been withdrawn from the market. The currently available parenteral direct thrombin inhibitors include argatroban and bivalirudin. Argatroban is a synthetic direct thrombin inhibitor derived from L-arginine that forms a noncovalent bond with the thrombin active site. It is hepatically cleared and has a half-life of 45 minutes in the presence of normal hepatic function. The target PTT therapeutic range is a ratio of 1.5–2.5. Bivalirudin is a synthetic direct thrombin inhibitor composed of the thrombin active site and heparin-binding site moieties of hirudin linked by a polypeptide linker. It is eliminated by plasma hydrolysis (90%) and renal clearance (10%) and has a half-life of 25 minutes in the presence of normal renal function. Argatroban and bivalirudin are FDA approved for treatment of HIT. 97,112

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Acquired Coagulation Disorders Caused by Inhibitors

Rebecca Kruse-Jarres MD, MPH , Cindy A. Leissinger MD , in Consultative Hemostasis and Thrombosis (Third Edition), 2013

Fibrinogen Inhibitors

Acquired antibodies that cause bleeding either by neutralizing functional fibrinogen or interfering with fibrin formation or polymerization are exceedingly rare, with fewer than 10 cases reported in the literature. Laboratory findings have usually included an elevated thrombin time and reptilase time, sometimes with normal levels of fibrinogen. Laboratory evidence in support of an inhibitor comes from mixing studies that show prolongation of the thrombin time in control plasma after mixing with patient plasma. In several cases, the inhibitor was shown to be an IgG autoantibody. 112-114

Female patients appear to be the most commonly affected. Of three teenaged girls affected by these inhibitors who were described in separate reports, two patients had an underlying autoimmune disorder 113,114 and symptoms in the other girl followed a gastrointestinal illness. 115 Another adult female developed an inhibitor while taking isoniazid, which rapidly disappeared after the drug was stopped. 112 Bleeding symptoms at presentation included ecchymoses, menorrhagia, and gastrointestinal bleeding. Little information is available on treatment of bleeding in these patients. The inhibitor spontaneously remitted in at least two cases but persisted in several patients with underlying lupus or other autoimmune disorders.

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Burn Wound Management

Katie Osborn , in Global Reconstructive Surgery, 2019

Skin Grafting

Burn wounds are closed using either split-thickness skin grafts or, in small areas, full-thickness skin grafts. The grafts are harvested from a donor site of the patient's non-burned, intact skin. When the split-thickness graft is applied to the wound as harvested, it is referred to as a sheet graft (see Fig. 4.2.7A). An alternative method of grafting is to mesh the graft (cutting small holes in it), which allows the skin to be stretched over a greater surface area (Figs. 4.2.8A and B). Meshing can increase the graft to several times its original size, depending on the size of the holes. The holes will close over time by epithelialization from the skin edges. Fig. 4.2.8D is a healed meshed skin graft.

Eventually, both sheet and meshed skin grafts adhere to the wound by fibrin formation. Circulation to the graft begins within 48 hours, as evidenced by an increasing pink or red color of the grafted area. Grafts may be sutured or stapled in place, although an essential part of nursing care is to protect skin grafts until healed. Loss of the skin graft may occur for several reasons including:

•

Hematoma and seroma formation under a sheet graft will prevent the graft from adhering to the wound surface. Therefore during the first few days after surgery, sheet grafts must be examined regularly, and hematomas and seromas must be removed either by needle aspiration, incising the graft to express blood and fluid, or rolling the fluid to the side of the graft with a cotton-tipped applicator. Meshed split-thickness skin grafts are less likely to have blood and serous fluid collect under the grafted area. Nevertheless, monitoring for fluid accumulation is still indicated.

•

Motion of the grafts will prevent vascular connections and graft adherence. The graft must remain immobilized. Splinting the grafted area is an essential aspect of nursing care, along with educating the patient and family about the importance of immobilization. Normal activity can usually be resumed in about 5 days, depending on the preference of the surgeon and the condition of the grafted area.

•

Infection in the grafted area will prevent graft adherence. 8 Strict aseptic technique when caring for the skin grafts will help prevent infection.

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