Silverstein and Hopper Chapter 101: Hypercoagulable States

Derangements in the Coagulation System that Tip the Balance in Favor of Coagulation

Increased procoagulant elements

Decreased anticoagulants

Diminished fibrinolysis

What causes thrombophilia?

Inherited conditions

• In people include factor V Leiden mutation and protein C deficiency

• No inherited forms of thrombophilia described in veterinary medicine

Acquired causes

What comprises Virchow’s triad?

Endothelial dysfunction

Hypercoagulability of blood

Blood stasis or altered blood flow

Phenotype of the Endothelium in Heath and During Injury

• In health, the endothelium exhibits an anticoagulant phenotype, maintaining normal blood flow and organ perfusion

• Upon activation or injury, the endothelium transitions to a prothrombotic phenotype

What can activate vascular endothelial cells?

TNF-a, bradykinin, thrombin, histamine, and vascular endothelial growth factor

What happens once endothelial cells are activated?

• Once activated or injured, ECs release ultra-large multimers of vWF (UL-vWF) from the Weibel-Palade bodies

  • UL-vWF can bind platelet GP Iba receptors and initiate platelet tethering and activation

    • More active for platelet adhesion and activation than smaller vWF multimers

  • In health, UL-vWF are quickly cleaved into smaller multimers by a disintegrin-like and metalloproteinase with thrombospondin type 1 repeats (ADAMTS13)

    • These smaller vWF molecules circulate freely in association with FVIII and have considerably less platelet aggregatory activity than the UL-vWF molecules

    • UL-vWFs usually remain tethered at sites of endothelial activation or injury, bound to the cell surface or to exposed collagen

    • Decrease or absence of ADAMTS13 may result in high concentrations of UL-vWF -> systemic platelet aggregation, thrombosis, and a subsequent consumptive thrombocytopenia

What percentage of the proteglycans in the glycocalyx does heparin sulfate account for?

50-90%

What is action of heparin suflate?

Facilitates the binding of antithrombin (AT), which increases the efficiency of AT-mediated inhibition of thrombin

What are the roles of the luminal glycocalyx in anticoagulation?

• Composed of a large network of negatively charged glycosaminoglycans (GAGs), proteoglycans, and glycoproteins

• Other important anticoagulants bind the glycocalyx, including heparin cofactor II and TM

• Tissue factor pathway inhibitor (TFPI) localizes to the glycocalyx

• Glycocalyx also serves as a mechanoreceptor, sensing altered blood flow and releasing NO during conditions of increased shear stress to maintain appropriate organ perfusion

• With inflammation there is decreased synthesis of the GAGs that comprise the glycocalyx, decreasing the function of the anticoagulants that rely on the glycocalyx (TM and protein C, TFPI)

• Glycocalyx buffers ECs by preventing the binding of inflammatory cytokines to cell surface receptors

Roles of Tissue Factor in Coagulation

• Endothelial disruption exposes procoagulant substances such as tissue factor (TF) to the circulating blood

  • Coagulation in vivo is initiated by interaction of TF with activated factor VII

  • TF may be expressed on monocytes/macrophages that have been activated by inflammation and it has been identified on the surface of neoplastic cells

  • TF perpetuates inflammation through the activation of nuclear factor kB, leading to the production of TNF-a

What happens once platelets are activated?

• Upon activation, platelets undergo a shape change and shuffle negatively charged phospholipids (phosphatidylserine and phosphatidylethanolamine) to the surface

  • These provide the catalytic surfaces essential for the tenase and prothrombinase complexes for the propagation phase of clot formation

  • With activation, platelets activate and increase the number of copies of the active fibrinogen receptor (GP IIbIIIa or integrin aIIbB3) on their surface

  • The contents of alpha and dense granules are secreted, releasing procoagulant elements like calcium, factor Va, serotonin, fibrinogen, P-selectin, and AP

Microparticles

• Circulating small vesicles released from activated or apoptotic cells

  • May be derived from platelets, ECs, leukocytes, erythrocytes, and neoplastic cells

  • Can provide an asymmetric phospholipid membrane for thrombin generation

  • MPs can express TF on their surface

    • Those expressing both phosphatidylserine and TF are characterized as procoagulant MPs

      • Other procoagulant MPs may display vWF-binding sites and UL-vWF multimers which can tether and activate circulating platelets

Roles of Neutrophil Extracellular Traps (NETs)

• Released from activated neutrophils and consist of nuclear contents, including DNA, histones, and some extranuclear proteins

• Role of NETs is immobilization and sequestration of microbial elements as a response to septicemia

• Also released following interaction with pathogen-associated molecular patterns

• In addition to antimicrobial properties, NETs can be procoagulant, specifically through activation of the contact pathway and potentially through direct platelet activation

• May impair fibrinolysis

What are the three main anticoagulant proteins?

Antithrombin (AT)

Protein C

Tissue factor platelet inhibitor (TFPI)

What are the actions of antithrombin?

• Primarily acts to inhibit thrombin and factor Xa

• Has lesser inhibitory effects on factors Ixa and the fVIIa-TF complex

When is antithrombin most effective?

When bound to heparin-like GAGs of the glycocalyx (e.g. heparan sulfate) or when exposed to endogenous heparins, increasing the inhibition of thromb greater than 1000-fold from nonbound AT

What are the mechanisms that decrease antithrombin in systemic inflammation or critical illness?

Consumption (due to thrombin generation)

Decreased production (negative acute phase protein)

Degradation by NE

What is the action of Protein C?

Inhibits factors Va and VIIIa

When is protein C activated?

When trace amounts of thrombin binds thrombomodulin (TM) on the endothelium of the circulation

• Reaction is accelerated in the presence of the endothelial protein C receptor (EPCR)

What accelerates the action of Protein C?

In the presence of the cofactor protein S, activated protein C’s (APC) inhibition of Va and VIIIa is accelerated nearly 20 fold

Action of Thrombomodulin

By binding thrombin, TM helps generate APC and also prevents thrombin from acting on fibrinogen and platelets

• Reaction also generates thrombin-activatable fibrinolysis inhibitor (TAFI) which inhibits fibrinolysis

What makes the Protein C system less functional during systemic inflammation?

• Protein C system is less functional during systemic inflammation due to decreased hepatic synthesis of proteins C and S

  • Activation of protein C is also hindered by the effects of inflammatory cytokines on the endothelium and TM

    • TNF-a can downregulate the expression of TM

    • Elastase from endotoxin-activated neutrophils can cleave TM from the endothelium

      • Circulating or soluble TM is less effective than when it is complexed with the EPCR on the endothelium

        • Soluble TM is increased in people with sepsis and independently predicts the presence of DIC, MODS, and mortality

Where is tissue factor platelet inhibitor released from?

Primarily endothelial cells

Action of TFPI

Acts to inhibit FVIIa-TF complexes and factor Xa (all components of the TF or extrinisc pathway)

Protein S serves as a cofactor for the inhibition of FXa by TFPI

How does fibrinolysis normally work?

• Fibrinolysis is the final protective step to prevent vascular occlusion

• Circulating plasminogen is incorporated into forming clots

  • Converted to plasmin by fibrinolytic activators (tPA and urokinase)

    • tPA and urokinase are derived largely from the endothelium and released upon activation or injury

  • Plasmin breaks down the fibrin meshwork of the formed clot and allows for recannulation of blood vessels

What decreases fibrinolysis?

• Effects of plasminogen are decreased by endogenous plasminogen activator inhibitor (PAI-1)

  • In the presence of TNF-a and IL-1B, there is a delayed but more sustained increase in PAI-1 than tPA, decreasing fibrinolysis and resulting in the persistence of thrombi

• NETs contribute to delayed fibrinolysis through integration into clots (making the clots more resistant to fibrinolysis) and supporting the activity of PAI-1

What decreases the effects of plasminogen?

Plasminogen activator inhibitor (PAI-1)

What is one of the only means for a clinician to definitively learn that pathologic coagulation is occurring?

Detecting the presence of a thrombus or thromboembolus

Traditional Coagulation Tests for Hypercoagulability

Prolongations of aPTT/PT and decreased platelet count may appear in patients with hypercoagulability, but this is usually due to consumption of platelets and coagulation factors following unregulated thrombin generation

• A drop in circulating platelet count accompanied by a 20% or greater prolongation in baseline aPTT in an at-risk patient should raise concern of a consumptive coagulopathy

What does documentation of a hypercoagulable state rely on?

• Either on identifying a rise in procoagulant elements (e.g. MPs, fV or VIII activities, or fibrinogen), a decrease in endogenous anticoagulants (e.g. AT, protein S and C, or TFPI), or a decrease in fibrinolysis (e.g. decreased tPA, increased a2-antiplasmin, PAI-1, TAFI)

  • Markers of ongoing thrombin generation (e.g. thrombin-AT complex [TAT], prothrombin activation fragment [F1+2], or fibrinopeptides A and B) or lysis of fibrin clots (fibrinogen degradation product [FDP] or D-dimer) may be used

What does viscoelastic testing evaluate?

The time to initial fibrin crosslinking, rate of clot formation, and the viscoelastic characteristics of the clot formed

What does CAT focus on?

The thrombin generation potential (endogenous thrombin potential, ETP) in a sample

How can platelet contribution to a hypercoagulable state be inferred?

By assessing markers of platelet activation (e.g. P-selectin expression, platelet-neutrophil aggregates) or documentation of hyperfunctional platelets in response to standard stimuli

Mean Platelet Component (MPC)

Related to the granularity of the circulating platelets

• Following activation, the granularity of platelets decreases

• A decreased MPC may represent circulating activated platelets

CURATIVE Recommendations for IMHA (Dogs)

IMHA is strongly associated with the development of thrombosis

Antithrombotics are recommended

CURATIVE Recommendations for PLN

PLN is associated with the development of thrombosis

Antithrombotics are recommended

CURATIVE Recommendationsn for Cardiomyopathy (Cats)

Feline cardiomyopathy is strongly associated with a risk of arterial thromboembolism (ATE)

Cats with prior ATE, spontaneous echocontrast, or reduced left atrial appendage flow velocity may be at particular risk

Antithrombotic therapy is recommended, particularly when these risk factors are present

CURATIVE Recommendations for Pancreatitis (Dogs)

Severe pancreatitis, in particular acute necrotizing pancreatitis, may be associated with the development of thrombosis

CURATIVE Recommendations for Neoplasia

Cancer in dogs, in particular (adeno)carcinoma, is associated with the development of thrombosis in a small subset only

There is insufficient evidence to support routine anticoagulation with cancer, but antithrombotic therapy should be considered where hypercoagulability is demonstrated or other risk factors exist

CURATIVE Recommendationsfor Hypercortisolemia (Dogs)

Corticosteroid administration favors a hypercoagulable state and thus may be associated with the development of thrombosis, particularly in patients with other risk factors for thrombosis

• Antithrombotics should be considered where other risk factors exist

Natural hyperadrenocorticism (HAC) is associated with a risk of thrombosis in a small subset of dogs only. HAC alone doesn’t warrant therapy in the majority of dogs, unless other risk factors are present

CURATIVE Recommendations for Sepsis (Dogs)

Sepsis is associated with the development of thrombosis in a small subset only

There is insufficient evidence to support routine anticoagulation; however, antithrombotics should be considered where hypercoagulability is demonstrated, or when other risk factors are present

CURATIVE Recommendations for Cerebrovascular Disease

Cerebrovascular disease is more likely the result of a thrombotic event, rather than the cause

Antithrombotic therapy should be considered when an ischemic stroke is identified and a concurrent condition associated with a risk of thrombosis is present

CURATIVE Recommendations of Antithrombotic Therapy Following Dissolution of an in situ Blood Clot

When an underlying condition is unknown or cannot be cured, antithrombotics should be continued indefinitely

When a causative condition is resolved, antithrombotic therapies should be discontinued following thrombus resolution

Low or moderate risk patients: the potential adverse effects associated with risk of discontinuation should be weighed against the risk of recurrent prothrombotic conditions

Coagulopathy Associated with Systemic Inflammation

• Increased expression of TF

• Activation of ECs and disruption of the glycocalyx

  • Leads to compromised production of local regulators (e.g. NO) and increased expression of procoagulant molecules (e.g. UL-vWF or TF) and adhesion molecules (e.g. P-selectin) with derangement of anticoagulant defenses

  • TM may be damaged by multiple mechanisms (leading to decreased activation of protein C) and AT is less effective due to both decreased concentrations and impaired interactions with an endothelium that has been denuded of GAGs

  • TFPI may have impaired EC localization

• Impairment of anticoagulant systems

• Abatement of fibrinolysis

  • Exuberant release of PAI-1 due to inflammatory cytokine release can slow fibrinolysis and further impede coagulation defenses

• Patients with sepsis develop an initial hypercoagulable phase, followed by a much longer hypocoagulable phase due to consumption

  • Majority of patients described in the veterinary literature display a hypocoagulable phenotype with evidence of prior clot formation

Coagulopathy Associated with Protein Losing Nephropathy

• In people, the thrombophilia associated with PLN appears to be multifactorial

  • Platelets are hyperaggregable and exhibit increased markers of activation (e.g. P-selectin)

  • There are often increases in fVIII activity and fibrinogen concentration, while vWF levels and fV are variably elevated

  • Loss of endogenous anticoagulant potential centers on low AT activity

    • AT activity fails to uniformly predict thrombotic risk across studies in people

  • People have a propensity to develop renal vein thrombosis and increased markers of endothelial activation have been documented suggesting some involvement of a local mechanism (e.g. endothelial activation or abnormal renal blood flow) contributing to the overall thrombophilia

Coagulopathy Associated with Immune-Mediated Hemolytic Anemia

• Thrombi have been identified in up to 46-80% of nonsurvivors and DIC in 45% of dogs suffering from IMHA

• The majority of deaths with IMHA occur within the first 2 weeks, largely due to anemia and/or thrombotic complications

• Coagulation abnormalities consistent with a hypercoagulable state (low AT activity, elevated FDPs and D-dimer, and markedly elevated fibrinogen concentration) are commonly reported in this population

• TEG studies have also documented hypercoagulability, primarily on the bases of an increased clot strength (maximal amplitude [MA])

  • Fibrinogen, platelet count and function, and hematocrit are all key contributors to the MA

• Circulating TF is also a likely contributor to the procoagulant sate of IMHA with upregulation of TF gene expression present in whole blood

• Dogs with IMHA have increased concentrations of circulating cfDNA, partially due to NET formation from neutrophil responses to hypoxia, exposure to free heme, or inflammation

Coagulopathies Associated with Hypercortisolemia

• Hyperadrenocorticism (HAC) in people is associated with an increased risk of thrombotic complications

  • Changes identified in people include elevated activities of fVIII and vWF, heightened levels of PAI-1, and elevated activities of factors IX, XI, and XII

• A consistent cause or definable procoagulant state has not been identified in dogs

Coagulopathies Associated with Cardiomyopathies

• Arterial thromboembolism (ATE) in cats is most commonly associated with cardiac disease

• Left atrial (LA) and LA appendage enlargement are associated with numerous structural changes, culminating in a procoagulant phenotype, such as increased TF and vWF on areas of denuded or damaged endothelium

• Through atrial enlargement, shear stress is decreased (stasis), reducing the release of NO

Coagulopathies Associated with Neoplasia

• DIC has been described in 9.6% of dogs with malignancies with the highest rates occurring in dogs with hemangiosarcoma, mammary carcinoma, and adenocarcinoma of the lung

• The components of coagulation are not only contributors to thrombosis, but important in cancer behavior, in particular tumor growth, angiogenesis, and metastasis

• TF has been identified on malignant cells and in the tumor vasculature and these cells have the ability to shed TF-bearing MPs

  • TF expression on histopathology samples is an independent predictor of poor overall or relapse-free survival in many tumor types in people

• Patients with distant metastasis commonly have higher fibrinogen and D-dimer levels than locally invasive or noninvasive disease

How do you manage hypercoagulable conditions?

Treatment of the underlying condition

Antithrombotic therapy

What are options for antithrombotic therapy?

• Can consist of drugs that inhibit platelet function (e.g. aspirin or clopidogrel) or drugs that facilitate the inhibition of thrombin (e.g. unfractionated heparin (UFH) or low molecular weight heparin [LMWH])

• Antithrombotics should be used when a patient has an identified risk for thrombotic complications and the risks of thrombosis outweigh possible adverse effects of the therapy

• Oral direct factor Xa inhibitors (e.g. apixaban or rivaroxaban) are gaining popularity in veterinary medicine

Antithrombotic Therapy for Inflammatory Conditions

• Coagulation testing should be evaluated frequently with particular attention paid to those exhibiting more than one significant predisposition

• A drop in platelet count or 20% or greater prolongation in aPTT should raise concerns for the early stages of a consumptive coagulopathy

• Difficult to slow consumption of coagulation components once DIC is initiated

• High-dose heparin therapy after initiation of a consumptive coagulopathy might worsen the clinical picture and the identification of the hypercoagulable phase when heparin therapy may be the most useful remains difficult

Antithrombotic Therapy for Protein Losing Nephropathy

• Any patient with PLN or NS, and likely those with significant proteinuria may benefit from some form of thromboprophylaxis

• Historically have been treated with platelet inhibitors such as aspirin

• In cases of markedly decreased AT activity, heparins may have less efficacy and other anticoagulants (e.g. direct factor Xa inhibitors) may be needed for more substantial anticoagulation

Antithrombotic Therapy for Immune-Mediate Hemolytic Anemia

• In one retrospective study, aspirin was associated with an increased survival benefit

• In a study, dogs receiving clopidogrel + aspirin did not develop any apparent thrombotic tissues

Antithrombotic Therapies for Hypercortisolemia

• Testing for markers to support a prothrombotic state seems prudent prior to anticoagulation

Antithrombotic Therapies for Cardiomyopathies

• Long-term thromboprophylaxis in cats with cardiomyopathies has traditionally been with an oral platelet inhibitor

• Thrombolysis may be considered for any recent onset ATE with signs of ischemia

  • Studies of cats undergoing thrombolysis suggest a similar survival compared with conservative management with a heightened risk of complications

Antithrombotic Therapies for Neoplasia

• Tumors of epithelial cell origin and hemic neoplasms (e.g. lymphoma, leukemia, histiocytic sarcoma, hemangiosarcoma) are among the most commonly implicated tumor types in thrombotic complications

• Any cancer may promote thrombus formation due to alterations in vascular flow, endothelial damage, inflammation, or a combination of all three

• Various antithrombotics (e.g. warfarin and aspirin) have been implicated in decreasing the rate of metastasis with cancer, presumably by preventing metastasis on thrombi, activated platelets, or possibly MPs

• Coagulation testing is advised in patients with known predilections, especially in those with greater tumor burdens

• Antithrombotics should be considered when testing suggests a risk for thrombin generation (e.g. elevated D-dimer or TAT) in concert with a clinical suspicion