Physiology of normal hemostasis

Hemostasis dependent on both cellular components and soluble plasma proteins is the physiologic mechanism that halts bleeding after injury to the vasculature. Circulating platelets adhere and aggregate at sites of blood vessel injury. Platelet adhesion is dependent on the presence of the von Willebrand factor (vWF) and is followed by an aggregation response. Activation of platelets results in a flipping of the polarity of inner and outer leaflets of the cytoplasmic membrane and consequent exposure of anionic phospholipids that serve as platforms for the assembly of blood coagulation enzyme complexes. The extrinsic pathway of blood coagulation is initiated when blood is exposed to tissue factor (TF), a transmembrane protein expressed in the deeper portions of the blood vessel wall that may also be present in stimulated endothelial cells. Thrombus propagation occurs via incorporation of active, blood-borne TF into the growing clot.⁶ TF binds activated factor VII (factor VIIa) and the resulting complex activates factors X and IX. Activated factor IX (factor IXa) combines with factor VIIIa to provide a second pathway to activate factor X. Factor Xa complexes with factor Va and prothrombin to form prothrombinase, which cleaves prothrombin to generate thrombin, the key enzyme in hemostasis. In the final step of the coagulation cascade, thrombin cleaves fibrinogen to generate fibrin monomers, which then polymerize. This polymer is covalently cross-linked by factor XIIIa (itself generated from factor XIII by thrombin) to form a chemically stable clot. Thrombin also feeds back to activate cofactors V, VIII, and XI further amplifying the coagulation system.⁷

Fibrin deposition is limited by an endogenous anticoagulant system. Antithrombin (AT) is a plasma protein member of the serpin (serine protease inhibitor) family that inhibits the activities of all of the activated coagulation enzymes particularly factors IIa and Xa. Protein C is a vitamin K-dependent protein that proteolyzes factors Va and VIIIa to inactive fragments. Protein C binds to an endothelial cell protein C receptor (EPCR)⁸ and is activated by thrombin bound to thrombomodulin, another endothelial cell membrane-based protein, in a reaction that is modulated by a cofactor, protein S. TF pathway inhibitor is a plasma protein that forms a quaternary complex with TF, factor VIIa, and factor Xa, thereby inhibiting the extrinsic coagulation pathway.

There has been increasing intriguing evidence that, in addition to its role in hemostasis, factor XI plays a role in venous thrombosis. This has been supported by a recent clinical trial of a factor XI antisense oligonucleotide in patients who were at increased risk for DVT because of knee replacement surgery in which this strategy significantly reduced venous thrombosis. This type of anticoagulant strategy has the possibility of yielding important clinical benefits for cancer patients as it appears that factor XI level did not need to be reduced below a homeostatic level of 20% in order to gain a prophylactic effect. It is therefore possible that this strategy may be able to reduce the rate of thrombosis without significantly increasing the risk of bleeding.⁹ However, this possibility will require testing in well-designed clinical trials.

The fibrinolytic system refers to a cascade of serine proteases that result in fibrin degradation and clot dissolution. The final step in this process is the conversion of the circulating zymogen plasminogen into its active form plasmin. In the circulation, this is mainly achieved by tissue-type plasminogen activator (t-PA). This catalysis is highly dependent on the presence of fibrin, as binding of both plasminogen and t-PA to fibrin increases plasmin generation by more than two orders of magnitude. Mechanistically, plasminogen binds to exposed lysine residues formed in fibrin and these binding sites increase in number during fibrin cleavage allowing more plasminogen binding to occur, thereby amplifying the process allowing more plasmin to be generated. Naturally occurring plasma inhibitors [α2 antiplasmin, plasminogen activator inhibitor (PAI)-1, and PAI-2] also exist to limit plasmin activity or its generation in the circulation. The most potent of these is α2 antiplasmin. Plasmin is largely protected from α2 antiplasmin while bound to fibrin, allowing fibrin cleavage to occur. Other key regulatory steps occur at the level of the plasminogen activators, as the activity of t-PA and also urokinase-type plasminogen activator (u-PA), the second important endogenous plasminogen activator, are both regulated by PAI-1 and PAI-2.

The most recently described mechanism that limits the fibrinolytic system is via “thrombin activatable fibrinolysis inhibitor.” The protein, thrombin activatable fibrinolysis inhibitor (TAFI), is a carboxypeptidase that specifically removes exposed lysine residues from fibrin, thereby removing the ability of plasminogen and t-PA to dock onto lysine binding sites in fibrin. As it requires activation by thrombin, TAFI becomes engaged as a direct consequence of coagulation to stabilize and protect clots from premature removal by the fibrinolytic system.¹⁰

Relationship of coagulation system, inflammation, and cancer

The relationship between blood coagulation and cancer was first described in the medical literature during the latter half of the nineteenth century with Trousseau’s classic report of the association of migratory thrombophlebitis and gastric carcinoma.¹¹ In 1878, Billroth demonstrated cancer cells within a thrombus and theorized that tumor cells were spread by thromboembolism.¹² The interaction between blood coagulation and tumor angiogenesis and growth is supported by the involvement of several coagulation factors, specifically TF and thrombin in cancer neoangiogenesis, growth, and dissemination. Cancer cells or host cells in response to the neoplastic process cause local and systemic inflammatory stimuli that can switch the endothelium to a prothrombotic surface.¹³ Endothelial damage leads to exposure of subendothelial VWF and TF. VWF in turn induces platelet and tumor cell adhesion, with subsequent platelet activation and aggregation. TF plays a key role in the initiation of the coagulation cascade. TF is aberrantly expressed on the surface of activated endothelial cells, monocytes, and tumor cells.¹⁴ TF is upregulated in endothelium in pathologic states such as cancer as evidenced by the expression of TF and cross-linked fibrin on the endothelium of newly formed blood vessels within human tumors.¹⁵

Microparticles

Cell-derived vesicles, in particular extracellular vesicles (EVs) such as microparticles (MPs) and microvesicles, besides exosomes, are raising more and more attention as a novel and unique approach to detecting diseases.¹⁶ MPs are generally defined as 0.1 to 1 µm membrane particles that expose the anionic phospholipid phosphatidylserine and membrane antigens representative of their cellular origin. Platelet-derived microparticles (PMPs) represent the most abundant MP subtype. Their presence reflects platelet activity, physiopathology, and the thrombotic state of cancer patients. Because platelets play a key role in cancer progression, as well as formation of metastasis, PMPs also may be important in the proliferation of cancer cells, cancer cell interactions, metastatic progression, angiogenesis, and inflammation.¹⁷

While this is of great research interest and has diagnostic potential, the methodology(ies) for MP analysis has not yet been sufficiently standardized for routine clinical use. The authors of a recent consensus workshop concluded that there was significant variability among laboratories even when a common protocol was utilized.¹⁸

Bleeding disorders

Cancer can cause both quantitative and qualitative changes in platelets.¹⁹ Reactive thrombocytosis occurs in approximately 60% of cancer patients, while thrombocytopenia occurs in up to 11% of patients with untreated malignancy (Table 1).

Elevated microparticles have increasingly been associated with thrombosis in cancer; however, these tests have not yet been sufficiently standardized to be useful.

DIC, disseminated intravascular coagulation; TMA, thrombotic microangiopathy; IL, interleukin; TNF, tumor necrosis factor.

The major bleeding problems are commonly caused by tumor invasion of blood vessels and adjacent organs, complications of treatment, and vitamin K deficiency. Bleeding in the cancer patient may present as either localized bleeding usually as a result of tumor invasion or as generalized bleeding diathesis caused by thrombocytopenia, thrombocytopathies, specific coagulation factor deficiencies, disseminated intravascular coagulation (DIC), or hyperfibrinolysis.¹⁹ Appropriate treatment of bleeding in cancer patients needs to address the underlying disorder responsible for the bleeding.

Coagulation factor deficiencies

Cancer patients may develop various coagulation factor abnormalities resulting from vitamin K deficiency as a consequence of malnutrition, diarrhea, liver disease, biliary obstruction, use of oral anticoagulants, and antibiotic therapy. Patients with primary or metastatic hepatocellular carcinoma have deficiency of vitamin K-dependent factors (factors II, VII, IX, and X and proteins C and S), similar to that seen with liver cirrhosis. These patients almost always have increased levels of fibrinogen, unlike patients with cirrhosis or acute liver failure who have decreased fibrinogen levels. Acquired inhibitors of coagulation factors are frequently seen in multiple myeloma and other plasma cell dyscrasias.

The treatment of cancer patients with coagulation factor deficiencies, aside from the treatment of the underlying neoplasm, is generally supportive, and consists of vitamin K, fresh-frozen plasma, and cryoprecipitate. Oral vitamin K is the treatment of choice. Cryoprecipitate is administered IV, generally at a dose of 1 unit of cryoprecipitate for every 5 kg of body weight.³⁷

Drug effects (L-asparaginase)

L-Asparaginase is used in combination with other agents, for induction of remissions in acute lymphocytic leukemia. L-Asparaginase can cause depletion of many of the coagulation factors with an associated risk for thrombosis and hemorrhage. Levels of t-PA and PAI were increased during treatment while cross-linked fibrin degradation products remained within normal limits, excluding the presence of DIC.³⁹

Acute promyelocytic leukemia

APL-associated hemostasis disorders result from at least two distinct mechanisms, the release of procoagulant activities and plasminogen activators from the leukemic cells. The T15–17 translocation induces hyperexpression of TF and renders the patient hypercoagulable. It appears that plasmin-dependent primary fibrinogenolysis is the major etiologic factor for low fibrinogen levels in APL patients. In vivo differentiation therapy with all-trans-retinoic acid induces a rapid decrease in plasmin activation and a normalization of fibrinogen level, and was associated with a significant decrease in TF gene expression in bone marrow cells.⁴⁰ Administration of heparin in APL has been discontinued in current treatment regimens that include ATRA. Heparin has been thought to control the coagulopathy associated with APL by inhibiting intravascular fibrin formation and reducing the consumption of clotting factors and platelets, thereby decreasing the bleeding tendency.¹

Thrombotic complications

The pathogenesis of thrombotic complications in cancer patients is multifactorial. In addition to the common predisposing factors for thrombosis such as immobility, venous stasis, advanced age, history of previous thrombosis, sepsis, and the use of central venous access devices, tumor cells have unique prothrombotic characteristics. Transformed malignant cells can induce platelet abnormalities, abnormal activation of the coagulation cascade, decreased hepatic synthesis of anticoagulant and coagulant proteins, fibrinolytic abnormalities, acquired thrombophilias, and expression of inflammatory and angiogenic cytokines (Table 1). Multiple biomarkers have been linked to cancer-associated thrombosis. The highest level of evidence currently exists for pre-chemotherapy-elevated platelets and leukocyte counts and low hemoglobin levels. D-Dimer is also predictive of cancer-associated VTE. Many cancer patients have elevated D-dimer levels without obvious thrombosis and there is no consensus on the cutoff levels that would be predictive of cancer-associated thrombosis. TF, the physiologic initiator of hemostasis, is widely expressed across many types of cancer. TF is released into the circulation in the form of MPs, and levels can be detected in cancer patients. There is no consensus “standard” TF assay however. Initial reports suggested a significant association of elevated TF with subsequent VTE. The majority of these data were derived from patients with specific cancers, particularly pancreatic cancer.

Several factors contribute to the increased risk for bleeding and thrombotic complications in the cancer patient (Table 4). Thrombotic manifestations in cancer patients may present as one of the following: migratory thrombophlebitis or Trousseau syndrome, venous thromboembolism (VTE), thrombotic microangiopathy (TMA), arterial thrombosis, and DIC.

^a Also bleeding.

Anticancer effects of anticoagulation treatment

Experimental and indirect clinical evidence suggests that anticoagulants, particularly LMWH, may have antineoplastic effects. It has been suggested that anticoagulants can interfere with tumor angiogenesis, proliferation potential of cancer cells, and the immune system by augmenting the antitumor activity of tumor necrosis factor and interferon mediated by NK cells.⁶⁹ Anticoagulants may also interfere with the various stages of the metastatic cascade.

Heparin-induced thrombocytopenia (HIT)

HIT is an immune-mediated thrombocytopenia that occurs in approximately 1–5% of patients receiving heparin.⁷⁰ The decrease in platelet count typically occurs 5–10 days after starting heparin but may develop within 24 h if there has been exposure to heparin during the preceding 3 months. Occasionally, the platelet count starts to fall only after heparin has been stopped (delayed-onset HIT). The frequency of HIT varies according to the type of heparin preparation (bovine UFH > porcine UFH > LMWH), the exposed patient population (postoperative > medical > pregnancy), and gender (female > male).⁷⁰ HIT is caused by heparin-dependent, platelet-activating antibodies that recognize platelet factor 4 (PF4) bound to heparin. The resulting platelet activation is associated with increased thrombin generation. Venous or arterial thromboses including deep venous thrombosis, PE, limb artery thrombosis, thrombotic stroke, and myocardial infarction can occur. A clinical pretest probability score known as the 4T’s (degree of thrombocytopenia, timing of thrombocytopenia, other etiologies of thrombocytopenia, and thrombosis) is useful in clinical practice. HIT should be suspected and treatment rapidly instituted in a patient with an intermediate of high test probability.⁷¹ In clinical practice, the laboratory diagnosis of HIT is made with a positive PF4-dependent immunoassay. Management consists of discontinuing all forms of heparin and using direct thrombin inhibitors such as lepirudin or argatroban, which do not have any cross-reactivity to HIT antibodies. For patients receiving warfarin at the time of diagnosis of HIT, reversal of warfarin anticoagulation with vitamin K is recommended.

Thrombotic microangiopathies

TMAs involve hemolytic anemia, thrombocytopenia, neurologic symptoms, renal dysfunction, and fever. The majority of these cases are reported in patients with adenocarcinoma, particularly gastric cancer; however, TMA also occurs in patients with breast cancer and lung cancer, and in Hodgkin and non-Hodgkin lymphomas.⁷² Thrombotic thrombocytopenic purpura/hemolytic uremic syndrome also occurs in association with cancer chemotherapy, especially with mitomycin C, bleomycin, cisplatin, and tamoxifen,⁷³ use of cyclosporine,⁷⁴ and interferon,⁷⁵ after hematopoeitic stem cell transplantation,⁷⁶ and with Sunitinib maleate, an oral multitargeting tyrosine kinase inhibitor approved for the treatment of metastatic renal cell carcinoma.⁷⁷

The pathophysiology of cancer-associated TMA is postulated to be similar to that of usual primary TMA. It involves injury to vascular endothelium with release of ultralarge vWF multimers due to a deficiency of a vWF-cleaving protease (ADAMTS-13) causing platelet aggregation.^{78, 79}

Typically, the microangiopathic hemolytic anemia and thrombocytopenia are severe and reticulocytosis is usually present, with increased levels of lactic acid dehydrogenase, reflecting intravascular hemolysis. The peripheral blood smear demonstrates numerous schistocytes. Renal failure and neurologic and pulmonary dysfunction are common. Standard treatment of TTP is plasmapheresis. Other treatment modalities that may be used in refractory cases include vincristine, intravenous gamma globulin, rituximab, and splenectomy.^80–82 Platelet transfusions are usually contraindicated because infused platelets may amplify the extent and severity of the formation of microvascular thrombi. Regardless of treatment, the prognosis of cancer patients with TMA is generally poor.

Arterial thrombosis and nonbacterial thrombotic endocarditis

The association between arterial thrombosis and cancer is less well described. Isolated cases have been reported and chemotherapy has been implicated as a cause.⁸³ The selective estrogen receptor modulators tamoxifen and raloxifene increase the risk of stroke,⁸⁴ especially in postmenopausal women at increased risk for coronary events and in current smokers.⁸⁵ Importantly, patients with acute ischemic stroke in the setting of active cancer (especially adenocarcinoma) have a significant short-term risk of recurrent ischemic stroke and other types of thromboembolism.⁸⁶

Nonbacterial thrombotic endocarditis (NBTE) represents a form of consumptive coagulopathy most commonly seen with adenocarcinomas of the lung and pancreas. The diagnosis should be suspected in any cancer patient who presents with ischemic embolic events. Echocardiography is diagnostic with the finding of sterile thrombotic vegetations on cardiac valves. In addition to valvular vegetations, ventricular segmental wall motion abnormalities resulting from silent embolization to the coronary arteries have been reported in 18% of cancer patients with NBTE. Management is essentially supportive and consists of treatment of the underlying cancer and anticoagulant therapy with unfractionated or low-molecular-weight heparin.

Disseminated intravascular coagulation (DIC)

DIC is a clinicopathological syndrome that complicates patients with malignancy. Patients with solid tumors (prostate, pancreas, lung, stomach, colon, and breast) and those with leukemia, especially APL, may be complicated by DIC that is manifested primarily by bleeding. Patients with hematologic malignancies often present with a state of chronic DIC in the absence of active thrombosis and/or bleeding. The bleeding disorder in APL is thought to be due to the abnormally high levels of expression of annexin A2 on APL cells, which leads to increased production of plasmin and ensuing bleeding from unopposed fibrinolysis. Annexin A2 is a phospholipid-binding protein on the surface of endothelial cells that serves to bind plasminogen and its activator, tPA. In APL cells, the t_15–17 translocation induces hyperexpression of TF in the leukemic cell, linking the primary oncogenic event with induction of hypercoagulability.⁸⁷

Plasma MP-associated TF procoagulant activity may play an important pathogenic role in the evolution of overt DIC in various types of malignancy.⁸⁸

The thrombotic disorders associated with DIC include recurrent venous thrombosis, peripheral arterial thrombosis, cerebrovascular thrombosis, disseminated arterial disease with organ failure, peripheral limb ischemia, and gangrene. Chronic forms of DIC are characterized by less florid clinical findings and more subtle, but persistent, laboratory abnormalities. Metastatic cancer is a common cause of chronic DIC. Over time, approximately 25% of patients with metastatic cancer develop a thrombotic event. In cancer patients, the diagnosis of DIC is made clinically and corroborated by a constellation of laboratory abnormalities (Table 6).⁸⁹ There is no single laboratory test that can establish or exclude the diagnosis of DIC. In most cases, a combination of tests in a patient with a clinical condition that is associated with DIC can be used to diagnose the disorder with reasonable certainty. In the presence of an underlying disease associated with DIC, an initial platelet count of less than 100,000/mm³ or a rapid decline in the platelet count and prolongation of the PT and aPTT is seen in about 50–60% of cases of DIC, and the presence of fibrin(ogen) degradation products and D-dimers in plasma. Fibrinogen levels may remain in the normal range in the face of its consumption because of increased synthesis of this acute-phase reactant. A finding of hypofibrinogenemia is only useful diagnostically in very severe cases of DIC. The peripheral blood smear may also demonstrate the presence of red cell fragmentation or schistocytes, but rarely >10% of the red cells. Soluble fibrin (SF) monomer, which is only generated intravascularly, is a sensitive but not specific test for the diagnosis of DIC.⁹⁰ There appears to be no added value measuring the natural anticoagulant protein C and/or AT.⁹¹ The International Society of Thrombosis and Hemostasis (ISTH) sub-committee of the Scientific and Standardization Committee (SCC) on DIC has recommended the use of scoring system for overt DIC.⁹¹ The sensitivity and specificity of the ISTH overt DIC score are 91% and 97%, respectively.⁹² It is important to repeat the tests to monitor the dynamically changing scenario based on the laboratory results and clinical manifestations.

^a Fibrinogen levels may remain in the normal range despite consumption because of increased production: a finding of hypofibrinogenemia is only useful diagnostically in very severe cases of DIC.

^b Factor VIII levels may be increased in some patients with early DIC because of thrombin activation of factor VIII.

^c Plasma D-dimers are specific cross-linked fibrin derivative generated when the endogenous fibrinolytic system degrades fibrin.

In general, the treatment of DIC is directed against the underlying cancer but supportive management to the bleeding or thrombotic manifestations is required. Cancer patients with DIC who are bleeding or at high risk for bleeding (patients undergoing surgery or invasive procedures) should receive platelet transfusions to maintain the platelet count greater than 50,000/μL and FFP (initial doses of 15 mL/kg, although a dose of 30 mL/kg produces a more complete correction of coagulation factor levels) if the PT or aPTT is prolonged. The administration of purified coagulation factor concentrates in DIC is not generally recommended unless patients are fluid overloaded and cannot receive FFP. Coagulation factor concentrates contain only specific factors, whereas in DIC, there is a global deficiency in coagulation factors. Severe hypofibrinogenemia (<1 g/L) needs to be treated with cryoprecipitate or fibrinogen concentrates if available. A dose of 3 g would raise plasma fibrinogen by 1 g/L, this can be given as two cryoprecipitate pools (10 donor units) or as 3 g of a fibrinogen concentrate. The response to the supportive transfusion therapy should be monitored clinically and with laboratory tests.⁸⁹ The bleeding associated with DIC in APL oftentimes responds dramatically to treatment with all-trans-retinoic acid.⁸⁶

Although there are no clinical randomized controlled trials demonstrating that the use of heparin in patients with DIC results in improved clinical outcome, intravenous heparin may be used in cancer patients with DIC-associated thrombosis for stabilization while the cancer is being treated unless moderate to severe thrombocytopenia or bleeding is present. The recommended dose of heparin is 10 U/kg/h by continuous IV infusion without a loading dose. Monitoring aPTT may be complicated but monitoring for signs of bleeding is important. In critically ill, nonbleeding patients with DIC, pharmacological thromboprophylaxis with either unfractionated or low-molecular-weight heparin is recommended.⁸⁹ In general, patients with DIC should not be treated with antifibrinolytic agents. However, in patients with DIC and bleeding secondary to primary fibrinolysis (e.g., prostate cancer), the fibrinolytic inhibitor, epsilon aminocaproic acid, can be administered with an initial IV loading dose of 4–6 g over 1 h followed by an IV infusion of 1 g/h while monitoring the clinical response. The recommended oral dose of aminocaproic acid is 50–60 mg/kg every 4–6 h.⁹³ However, in those patients with a primary thrombotic presentation and secondary fibrinolysis, fibrinolytic inhibitors should be avoided until the thrombotic process is controlled.⁸⁹

Drugs for treatment of bleeding and thrombotic disorders

Direct oral anticoagulants (DOACs)

Oral direct factor Xa inhibitors such as rivaroxaban, apixaban, and edoxaban and oral direct thrombin inhibitors such as dabigatran are being used in clinical practice for prevention of stroke and systemic embolism in the setting of nonvalvular atrial fibrillation and for the prevention and treatment of VTE. In the context of cancer patients, current clinical practice guidelines all recommend the use of therapeutic doses of LMWHs for the initial and long-term treatment of cancer-related thrombosis. The use of vitamin K antagonists (VKA) is acceptable if LMWH is not available. For the long-term treatment of VTE in patients with cancer, LMWH compared with VKA reduces venous thromboembolic events but not mortality. The decision for a patient with cancer and VTE to start long-term LMWH versus oral anticoagulation should balance the benefits and harms and integrate the patient’s values and preferences for the important outcomes and alternative management strategies.

DOACs have been shown to be comparable to conventional therapy for the acute treatment of VTE but their efficacy and safety in cancer patients remain uncertain. Their use cannot be supported until trials comparing them with LMWH are available.^{97, 98}

Summary

Bleeding and thrombotic complications are common in cancer patients. Significant advances in the understanding of the interrelationship between cancer, blood coagulation, and tumor angiogenesis have occurred in recent years. Bleeding complications usually result from abnormalities in platelets or deficiency of coagulation factors and require specific blood or coagulation factor replacement. Thromboembolic events including deep venous thrombosis and PE are not only common but also serious complications seen in cancer patients. Advances in novel biomarkers, diagnostic imaging, and availability of newer anticoagulant agents have greatly facilitated the care of these patients. Ongoing research in the understanding of the various disturbances in hemostasis, application of innovative treatment modalities, and use of appropriate thromboprophylaxis in cancer patients should ultimately lead to decreased morbidity and improved survival.

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