Hemostasis and Disseminated Intravascular Coagulation

Overview of Hemostasis

Hemostasis is a complex but elegant process of forming blood clots to seal injured vessels and degrading such clots after they have served their purpose. The various reactions that comprise hemostasis are traditionally conceptionalized as having three distinct phases: primary hemostasis, secondary hemostasis, and fibrinolysis. Primary hemostasis involves the formation of a platelet plug. Secondary hemostasis begins with cross-linking of fibrin strands and stabilization of the platelet plug to form a mature clot. The degradation of the mature clot describes the process of fibrinolysis. While this over-simplified scheme is useful in the evaluation of coagulation, especially in relation to typical laboratory coagulation tests, it is important to realize that in vivo, coagulation likely proceeds in a different manner. More recently, a new "cell-based" model of coagulation has been proposed to better explain the process of coagulation that occurs in the body [1,2]. The purpose of this review is to outline the classical approach to coagulation, introduce newer concepts in coagulation, and describe perturbations in coagulation that may culminate in disseminated intravascular coagulation (DIC). A discussion of coagulation tests, their interpretation and rational therapy of coagulation disorders completes this overview of hemostasis.

Primary Hemostasis

The inciting events leading to activation of coagulation are endothelial damage and subsequent exposure of collagen on the subendothelial surface. One of the mechanisms by which platelets adhere to the subendothelium is mediated by von Willebrand factor (VWF). A key event is the bridging between exposed collagen and various platelet surface glycoprotein receptors via VWF. Following adhesion, platelets release substances including adenosine diphosphate (ADP), serotonin, and platelet activating factor (PAF), which serve to activate platelets. This is followed by aggregation forming the temporary hemostatic plug. The formation of this temporary plug is referred to as primary hemostasis and is degraded within minutes. Concomitant with platelet aggregation, platelets release a large number of procoagulants and clotting factors, providing an optimum environment for activating the coagulation cascade and, thereby, initiation of secondary hemostasis.

Secondary Hemostasis

Stabilization of the temporary platelet plug with cross-linked fibrin strands derived from serine proteases constitutes secondary hemostasis. Traditionally, this process was thought to be composed of intrinsic, extrinsic, and common pathways (Fig 4.1). Similarly to activation of primary hemostasis, an irregular vascular surface is the contact stimulus necessary for activating factor XII into factor XIIa, which initiates the intrinsic (factors XI, IX, VIII) pathway. The extrinsic pathway is initiated by the release of tissue factor (TF), a transmembrane glycoprotein present in most non endothelial cell membranes (e.g., fibroblasts, vascular smooth muscle cells, monocytes). Tissue factor complexes with factor VII, and subsequently activates factors IX and X. Once factor X is activated, the common pathway of coagulation is initiated. Activated factors X and V culminate the conversion of prothrombin to thrombin, and finally thrombin converts fibrinogen into fibrin. Thrombin is also responsible for activating factor XIII which serves to cross-link fibrin strands and stabilize the clot, increasing its resistance to proteolytic degradation. Although the different pathways were once viewed as discrete events, it is now recognized that direct interactions occur between the pathways; the TF:factor VIIa complex (extrinsic pathway) also activates factors IX and X (intrinsic pathway). Nevertheless, the traditional scheme of secondary hemostasis offers a way to interpret common coagulation tests such as the prothrombin time (PT) and activated partial thromboplastin time (aPTT).

Figure 4.1 Classic coagulation cascade depicting reactions involved in the intrinsic, extrinsic, and common pathways, culminating in the production of the fibrin clot. HMWK = High molecular weight kalikrein, TF = tissue factor.

Fibrinolysis

Once the hemostatic plug has served its purpose it must be degraded and removed from the intravascular space. In addition to lysing fibrin and fibrinogen, plasmin biodegrades factors V, VIII, IX, and XI. Plasminogen, a proenzyme, is activated into plasmin by either factor XIIa or by a variety of poorly defined tissue factors. Several tissue activators of plasminogen have been recognized, including streptokinase, urokinase, and tissue plasminogen activator, and they may be used therapeutically in patients with thromboembolic disorders. The fibrinolytic system also has built-in inhibitory mechanisms that have a net procoagulant effect. Inhibitors of fibrinolysis include alpha-2 antiplasmin, alpha-2 macroglobulin and tissue plasminogen activator inhibitors 1 and 2. Plasmin biodegrades fibrinogen and fibrin-generating fibrin(ogen) degradation products (FDPs) that can be detected in plasma of dogs and cats. These FDPs also exert profound inhibitory effects on platelet function, contributing to the petechia and ecchymoses noted in patients with DIC.

New Model of Coagulation

Although the classic coagulation cascade is a convenient scheme that allows clinical assessment of coagulation status, it fails to explain why patients with severe congenital factor XII or XI deficiency do not exhibit significant bleeding disorders. In addition, it does not adequately explain why patients with deficiencies in factor VII but with intact intrinsic factors suffer from pronounced bleeding tendencies. It became apparent that coagulation in vivo must proceed differently from that demonstrated in vitro. A new model of coagulation emphasizes the role played by platelets and TF-bearing cells; hence it is referred to as the cell-based model of hemostasis (Fig 4.2). This model is described as having three phases: initiation, amplification, and propagation (Fig. 4-3).

Figure 4.2 Cell-based model of coagulation. Tissue factor (TF)-bearing cells first activate factor VII into VIIa. The TF:VIIa complex converts X and IX into Xa and IXa, respectively. Xa combines with Va and generates a small amount of thrombin, which activates platelets and other coagulation factors. The surface of activated platelets then becomes the site of major thrombin generation necessary for fibrin formation. vWF = von Willebrand factor, TF = tissue factor.

Figure 4.3. Stages of the cell-based model of coagulation. Initiation occurs on TF-bearing cells and results in the production of a small amount of thrombin. Thrombin produced by the initiation stage, activates platelets and other coagulation factors. Propagation occurs on the surface of activated platelets and results in the production of significant amounts of thrombin required for fibrin formation.

During the initiation phase, vascular injury exposes TF expressed on TF-bearing cells such as fibroblasts, smooth muscle cells, and monocytes. Factor VII becomes activated when complexed with TF. This complex proceeds to activate small amounts of factors IX and X. Activated factor X combines and activates factor V on the surface of the TF-bearing cell to produce a prothrombinase complex that generates thrombin. The small amount of thrombin generated is insufficient to catalyze fibrin generation and is limited to the vicinity of the TF-bearing cell. However, this small amount of thrombin serves as a priming mechanism for subsequent hemostatic events (see Fig. 4-2).

Platelet activation is central to the amplification phase, which sets the stage for subsequent large-scale generation of thrombin. The thrombin generated in the initiation phase stimulates release of factor V from circulating platelets, disengages factor VIII from VWF, and activates factors V, VIII, and XI on the platelet surface. Activated platelets also express receptors and binding sites for activated clotting factors. Uncoupled VWF can now mediate additional platelet adhesion and aggregation. The importance of the amplification phase is that platelets that were not directly stimulated by the inciting vessel-wall injury are recruited to participate in active coagulation.

The propagation phase is typified by the production of thrombin and fibrin from their inactive precursors (see Fig. 4-3). Activated factor XI produced in the amplification phase converts more factor IX into activated factor IX. Activated factor IX combines with its cofactor VIIIa to form the "tenase complex" (IXa/VIIIa). The tenase complex then recruits additional factor X from solution yielding more Xa on the surface of the platelet. Activated factor X, binds to activated factor V to form the "prothrombinase complex" that leads to a burst of thrombin generation of sufficient magnitude to clot fibrinogen and to the generation of the hemostatic plug.

Once the bleeding is abated, the clotting process must be limited to avoid thrombotic occlusion of adjacent normal areas of the vasculature. This may be thought of as a termination phase. The protein C/protein S/thrombomodulin system is an important mechanism confining coagulation to the site of injury [3]. Some of the thrombin generated during coagulation can diffuse away from the site of vascular injury. When thrombin reaches an intact endothelial cell, it binds to thrombomodulin (TM) expressed on the endothelial surface. The endothelial thrombin/TM complex then activates protein C, which binds to its cofactor protein S and inactivates any factors Va and VIIIa. This system illustrates the anticoagulant tendencies of the intact endothelium.

Disseminated Intravascular Coagulation

Rather than a disease, DIC is the pathologic consequence of many different disorders. It is characterized by an acute, generalized, widespread activation of coagulation, resulting in thromboembolic complications because of intravascular formation of fibrin, as well as diffuse hemorrhage, because of the consumption of platelets and coagulation factors [4-6]. Widespread fibrin deposition is associated with the genesis of multiple organ failure and poor outcome [4-8]. The clinical situation with simultaneous thrombosis and bleeding poses a difficult problem for the clinician in that replenishment of clotting factors with plasma does not attenuate the risk for thrombosis.

As DIC is always secondary to an underlying disorder, rational therapy should always be targeted at the primary disease. Common diseases associated with DIC are listed in Table 4-1. Disease processes characterized by capillary stasis, loss of vascular integrity, red cell hemolysis, inappropriate particulate matter in blood, or necrotic tissue release of tissue thromboplastin into the vasculature can produce this life-threatening complication.

The pathogenesis of DIC in patients with severe infections may involve exposure to specific cell membrane components of microorganisms (e.g., lipopolysaccharides) or bacterial exotoxins. A generalized systemic inflammatory response ensues, characterized by the elevation of several cytokines. Cytokines, produced mainly by activated mononuclear cells and endothelial cells are partially responsible for the derangement of the coagulation system in DIC [4-9]. Neutrophil activation leads to platelet activation and decrease in antithrombin activity. Disturbances in the antithrombogenic functions of the endothelium induce adherence of platelets and granulocytes through the expression of cell adhesion molecules, such as P-, E-, and L-selectins, endothelial leukocyte-adhesion molecules (ELAM), such as ELAM-1, and intercellular adhesion molecule-1 (ICAM-1) [4-8]. During this excessive intravascular coagulation, platelets are consumed in large quantities, causing thrombocytopenia. Tissue factor, which is exposed following injury to the endothelium and by activated monocytes, triggers the initiation of the coagulation cascade on the local endothelial surface. Local generation of thrombin leads to the formation of thrombi. In this process of self-perpetuating coagulation, coagulation factors are consumed. Once the fibrinolytic system is activated, inactivation of clotting factors, and impaired platelet function follows. Fibrin degradation products, produced by clot lysis, are strong inhibitors of platelet function. Antithrombin, along with proteins C and S, are soon depleted in attempts to halt intravascular coagulation. The fibrinolytic system becomes inhibited by the release of plasminogen activator inhibitor 1 (PAI-1). PAI-1 release is chiefly mediated by TNF, endotoxin, IL-1, and IL-6. This imbalance between the systems fosters fibrin deposition. The formation of fibrin within the microcirculation leads to hemolytic anemia as the red blood cells are sheared by the fibrin strands. As a result, fragmented red blood cells or schistocytes are found in circulation.

Assessment of Coagulation

Prothrombin time (PT) is the primary laboratory test to evaluate the status of the extrinsic pathway. For this test, blood is collected into tubes containing citrate, which binds calcium and halts coagulation. A thromboplastin reagent (source of TF) and calcium are added to initiate coagulation. The time to fibrin formation is measured in seconds and prolongation greater than 25% as compared with normal is considered significant. The PT is prolonged by deficiencies in factors II, V, VII, and X. Typically, prolongation in PT indicates that factor activity must be depleted to less than 30%.

The activated partial thromboplastin time (APTT) primarily reflects the integrity of the intrinsic pathway. As with measurement of PT, coagulation is halted by collecting blood into citrated tubes. An activator (e.g., kaolin, celite, propyl gallate) is added, supplying negatively charged particles similar to basement membranes to activate the contact group (i.e., factors XII, XI). Partial thromboplastin (source of phospholipids surfaces) and calcium are then added and the time to fibrin formation is recorded in seconds. Prolongations in APTT occur if factor activity of factors VIII, IX, X, XI, and XII decrease below 30%. Severe depression in factor activity of factors II and V and depletion of fibrinogen will also prolonged the APTT.

Activated clotting time (ACT) is a coarse assessment of the intrinsic pathway. Tubes containing diatomaceous earth provide a contact activator. Platelets supplied by whole blood, provide phospholipid membranes needed to support coagulation reaction. While normal reference ranges for PT and APTT depend on the particular reagents used, it is generally accepted that the normal ACT for dogs is 60 to 110 seconds and for cats 50 to 75 seconds.

Plasmin-mediated degradation of fibrinogen and fibrin produces several fragments (fragments X, Y, D, and E) collectively referred to as FDPs. The measurement of FDPs can be semi-quantitatively evaluated via agglutination techniques and is used as an indicator of active fibrinolysis. It is important to note that FDPs are produced from lysis of both cross-linked and non-cross-linked fibrin. As such, elevation in FDP concentration is merely an indicator of increased generation of fibrin and increased fibrinolysis. Kits to evaluate dimers of D fragments (D-dimers) are commercially available and are becoming more commonly used [10,11]. D-dimers are more specific for fibrinolysis (particularly of cross-linked fibrin), however, it does not distinguish between physiologic or pathologic fibrinolysis.

Diagnosis of DIC

The association between the development of DIC and poor outcome is the impetus for early diagnosis; however, therapy should be directed at the primary disease rather than at coagulation abnormalities. Despite the desire to identify afflicted patients, currently no consensus exists in definitively diagnosing DIC. Several criteria have been proposed, and many include abnormalities in any three coagulation parameters (prolongation in PT, APTT, decrease in platelet count, elevated FDPs or D-dimers, depleted fibrinogen or AT) [12,13]. In people, DIC scores and algorithms have also been proposed but are not widely recognized [6,14]. Various veterinary publications have each defined their criteria slightly differently, making development of similar schemes impossible [10,12,13]. Moreover, abnormalities in coagulation parameters are not specific enough for DIC and can be encountered in many disorders with vastly different prognoses.

In the CBC, schistocytes are indicative of mechanical damage to red cell membranes from microvascular fibrin strands. Whereas the presence of schistocytes is suggestive of DIC, they are only recognized in approximately 10% of patients with DIC [12,13]. Platelet counts can be variable in DIC because some inflammatory states can cause a reactive thrombocytosis and, therefore, it is more important to perform daily blood smears and manual platelet counts, as a drop in the platelet count often precedes other signs of DIC. In the future, increased platelet factor 4 and beta-thrombomodulin measurements could become pathognomonic for platelet destruction in DIC [4-7]. Dysfunctional platelets, resulting in thrombocytopathia, are caused by FDPs coating platelet membranes, which causes the release of platelet procoagulant materials.

Therapy for DIC

As DIC is a reflection of complete dysregulation of hemostasis, reversal can occur only if the primary stimulus is eliminated. However, as elimination of the initiating cause of DIC is almost never immediately possible, focus of therapy has traditionally been directed at halting further intravascular coagulation (administration of plasma, heparin, aspirin), promotion of capillary blood flow (aggressive fluid therapy), and supporting target organs at risk for hemorrhage, microthrombi, or ischemia (maintenance of perfusion and oxygenation, correction of acid-base status) [3-7]. Despite these generic recommendations, no studies exist supporting the use of such therapies at ameliorating or reversing DIC [7]. The most commonly employed therapies include administration of plasma and heparin, yet improvement in outcome has not been demonstrated in either people or animals with these approaches [2,15,16]. There is even a suggestion that while heparin does improve AT activity, it does so at the expense of potent anti-inflammatory properties of AT, making recommendations for heparin therapy questionable in patients with DIC [2,15,16]. In the future, strategies directed at inhibiting TF-mediated activation of coagulation or restoration of physiologic anticoagulant systems may prove beneficial. [7]

Conclusions

As hemostatic disorders are commonly encountered in critically ill patients, many of which require surgical intervention, an understanding of coagulation, its assessment, and relationship to disease pathogenesis is crucial for the clinician. Since surgical removal of tumors may be the most effective way to treat DIC, the presence of coagulation abnormalities alone does not preclude surgical interventions. Interpretation of coagulation tests must be done in the context of clinical situations so as to guide the most appropriate course of action. Discordant results of coagulation tests with clinical assessment of clinical or surgical bleeding may reflect differences between the processes of coagulation in vitro and those encountered in vivo. In spite of the progress in the understanding of coagulation, therapeutic decisions are still controversial and should be individualized on the basis and severity of the coagulation abnormality.