Abstract

DIC is a common complication of SIRS and sepsis. The development of DIC has also demonstrated to be a high predictor of mortality. SIRS and Sepsis are related to overproduction of proinflammatory mediators and acute phase proteins, systemic reproduction of pathogens and endotoxemia. If left unchecked by anti-inflammatory mechanisms, systemic inflammatory response damages the endothelial cells and activates coagulation by the expression of tissular factor TF, thrombomodulin consumption, diminished activation of protein C and its cofactor protein S., ATIII consumption and inactivation, deficiency of tissular factor pathway inhibitor TFPI and overproduction of plasminogen activator inhibitor PAI-1. Coagulation activation, inhibition of fibrinolysis and consumption of coagulation inhibitors leads to a procoagulant state resulting in inadequate fibrin removal and fibrin deposition in the microvasculature. Although the initial trigger and dynamics may vary, the clinical picture of severe sepsis or septic shock in latter stages is quite uniform. Fibrin deposition leads to diffuse obstruction of the microvascular bed resulting in tissular hypoperfusion, gastrointestinal barrier dysfunction, bacterial translocation, impaired phagocytic mononuclear system activity, increased risk for infection, progressive organ dysfunction (renal insufficiency, ARDS), increased systemic inflammatory response, hypotension and circulatory failure. In some cases, coagulation factor consumption and fibrin degradation products FDP may lead to diffuse bleeding.

Sepsis is a very common complication in critical patients and is generally considered an altered inflammatory response or SIRS. Disseminated intravascular coagulation (DIC) frequently complicates sepsis and has been diagnosed in 25-50% of septic human patients where it seems to be a strong predictor of mortality. Although the initial trigger and dynamics may vary, the hallmark of haemostatic disorders during sepsis is the imbalance between intravascular fibrin formation and its removal. Widespread fibrin deposition in the microvascular bed results in progressive ischemic organ damage and multiple organ dysfunction syndrome MODS. In some patients secondary fibrinolysis may lead to diffuse bleeding.

Coagulation--Inflammation That Malicious Vicious Cycle

Activation of endothelial cells is a main component in the pathogenesis of sepsis. Once activated by endotoxins, cytokines and excessive proinflammatory stimuli endothelial cells are capable of amplifying the inflammatory response, inducing cellular migration and the expression of adhesion molecules and protease receptors. Leukocytes produce free radicals, proteolytic enzymes like elastase and cytokines (TNF-α, IL-1, IL-6, etc.) that induce further endothelial damage. Exacerbated inflammatory response, immune mediated responses and necrosis additionally stimulates endothelial cells. The ischemic-reperfusion mechanism and acute-phase proteins like reactive protein C activate the complement and may induce local apoptosis thereby further propagating the inflammatory response. Coagulation activation during sepsis is primarily driven by tissue factor TF pathway. Damaged endothelial cells lose heparan sulphate and increase the synthesis of TF. After binding to exposed TF, circulating FVII is activated. The TF-FVIIa complex then activates FX to FXa which finally converts prothrombin to thrombin. It has been recently discovered that thrombin is capable of positively feeding the coagulation system by activating FV, FVIII and FXI in the common and intrinsic pathways, thereby greatly enhancing the capability of FXa to activate prothrombin. Moreover, TF-FVIIa complex can also activate FIX which in association with FVIIIa takes over the function of the extrinsic TF-FVIIa complex to activate FX, thus further propagating thrombin generation. Thrombin finally cleaves fibrinogen into fibrin monomers and activates FXIII that covalently cross-links fibrin monomers into a stable clot.

In a normal response thrombin generation by the TF-FVIIa-FXa complex is rapidly abrogated by the TF pathway inhibitor TFPI. 85% of the TFPI is bound via glycosaminoglycans to endothelial cells in the microvasculature; the rest circulates either associated with lipoproteins or platelets. Some studies have found normal or even elevated levels of TFPI in DIC and septic patients. However, during sepsis, there is also an over production of TF. Thrombin stimulates the production of platelet growing factor and it can also amplify the inflammatory response by its direct chemotactic properties. Activated monocytes, their released microparticles and damaged endothelial cells express TF thus producing a relative deficiency of TFPI to neutralize TF. The fibrinolytic system may be thereby overcome. During sepsis several anticoagulant mechanisms are severely impaired. Antithrombin AT is rapidly consumed as it forms thrombin-antithrombin complex T-AT, which are cleared from circulation. Moreover, neutrophils release elastase an enzyme that among others inactivates AT, and since AT is a negative acute-phase protein de novo hepatic synthesis is decreased during the acute phase of inflammation thus further compromising AT activity. Usually, thrombomodulin TM expressed on endothelial cells binds thrombin and activates protein

C. Activated protein C APC rapidly dissociates from the thrombin-TM complex and inactivates FVa and FVIIIa decreasing thrombin generation. However, during sepsis inflammatory mediators as TNF-α reduce the endothelial expression of TM, whereas the procoagulant response quickly consumes TM thereby reducing the production of APC. In addition, increased concentration of C4-binding protein inactivates protein S PS, which is required as an important cofactor of APC activity, thus further compromising its anticoagulatory activity. Fibrinolysis is activated during early phases of endothelial damage; apparently TNF-α is the main mediator of this activation. Circulant plasminogen is trapped in the fibrin net where it is converted into plasmin by several factors. The most important ones are tissue-type plasminogen activator (t-PA) which is produced by endothelial damaged cells and urokinase-like plasminogen activator. Plasmin is a potent proteolytic enzyme that digests fibrinogen and fibrin, thus producing fibrin and fibrinogen degradation products FDP as well as D-dimmer. Plasmin also digests prothrombin and factors V, VIII and XII thereby contraresting the spread of coagulation. Moreover, FDP may impair platelet activity and aggregation as well as thrombin activity and fibrin polymerization exerting anticoagulant properties. Plasmin is then rapidly inactivated by α₂-antiplasmin. However, during sepsis, fibrinolysis is attenuated by the increased endothelial production of plasminogen activator inhibitor PAI-1, which inactivates both t-PA and urokinase. Moreover, the amplified FXI-dependant thrombin formation can activate thrombin-activable fibrinolysis inhibitor, which cleaves off binding sites for plasminogen on fibrin, thus inhibiting fibrinolysis. The association of enhanced procoagulatory response and impaired fibrinolysis promote the widespread of microthrombosis inducing ischemic organ damage and MODS. Human patients with severe sepsis have strongly elevated PAI-1 levels, which have also demonstrated to be a strong predictor of mortality.

Anticoagulant - Antiinflammatory Effects the Key to Success

During the last decades studies performed in animal models as well as clinical trials in human patients with sepsis, have raised the hope that coagulation inhibitors that not only interfere with thrombin generation or action but also attenuate inflammation may reduce morbidity and mortality of severe sepsis.

APC is one of the main regulators of microcirculation and endothelial function, not only it has antithrombotic and profibrinolytic effects but also antiinflammatory activity. Some experimental models have shown that antiinflammatory effects happen even at lower levels than those required for anticoagulant effects. These effects are mediated by a specific endothelial PC receptor EPCR, which up regulates the expression of endothelial anti-apoptotic genes thereby reducing endothelial death and proinflammatory stimuli. Moreover, APC also modulates monocyte activation, inhibits TNF-α and IL-1 production and limits the expression of adhesion molecules, these effects seems to be independent from EPCR. Recombinant APC rAPC, at 24ug/kg/h CRI over 96hs, demonstrated a dose-dependent reduction in D-dimmer an IL-6 levels as well as a significant improvement in survival (19%). APC has a Grade B recommendation in the "Surviving Sepsis Campaign for management of severe sepsis and septic shock". To obtain optimal benefit avoiding bleeding complications, rAPC should only be administered in patients with platelets counts >30000/ul, an APACHE score > 25 with sepsis-induced MODS, septic shock or sepsis-induced ARDS.

High-dose treatment with purified as well as recombinant AT significantly reduced inflammatory response and mortality in experimental studies. Several randomized double blind studies in humans have demonstrated that in order to obtain a significant reduction over mortality, high AT levels should be achieved quickly after starting AT therapy.

In septic models in baboons, TFPI abrogated the procoagulant response, attenuated the inflammatory response and improved survival. Reduced mortality was also demonstrated in a peritonitis model in rabbits.

However, some discrepancies have been found in clinical trials in humans where no significant reduction and higher bleeding complications were found with TFPI and AT therapies, especially when associated with heparin. The probable explanation for that finding might be that the concomitant use of heparin decreases the ability of AT and TFPI to bind to glycosaminoglycans on endothelial cells thus increasing the anticoagulant properties but at the same time interfering with their antiinflammatory properties. In contrast, APC is the only inhibitor that can bind to endothelial cells independently of glycosaminoglycans through the specific EPCR.

Current recommendations emphasize the fact that AT substitution as a first-line therapy in septic patients concomitantly receiving heparin is not indicated. Nevertheless, on three human trials patients in the placebo group receiving prophylactic heparin had lower overall 28-day mortality. Moreover, the association of low doses of heparin with fresh frozen plasma FFP effectively reduced PAI-1 levels, renal fibrin deposition and mortality in a DIC model in rabbits. FFP has limited amounts of AT and therefore it is not an AT substitution therapy. Although, the role of low doses of heparin in septic patients requires further investigation, the conjunctive FFP-heparin therapy is still indicated in veterinary patients where AT substitution therapy may be unaffordable.

Early detection and treatment of DIC in septic patients is fundamental. The classic haemostatic profile for overt DIC includes thrombocytopenia, prolonged APTT and PT, reduced fibrinogen and ATIII, and increased D-dimmer and FDP. D-dimmer, FDP, APTT and ATIII are the most sensitive tests in dogs and horses. Cats may not show increased D-dimmer or FDP. The most important restriction of AT, TFPI or APC therapy in veterinary medicine is the elevated costs. However, the advances in recombinant technology and increased owner-demands for better treatments for their pets may transform these therapies in the routine for septic veterinary patients in the future.