Hypercoagulation During Sickle Cell Disease

Posted on: May 19, 2021

Hypercoagulation During Sickle Cell Disease

Contributed by Abi Kasberg, PhD

Sickle cell disease (SCD) is a genetic hemoglobin disease that disrupts the ability of red blood cells (RBCs) to deliver oxygen throughout the body. RBCs are normally round and flexible, which allows them to flow easily through vasculature. However, in SCD, RBCs are crescent-shaped due to the expression of an atypical sickle hemoglobin variant (HbS). The abnormal shape of sickle RBCs can cause them to clump together which blocks blood flow and promotes thrombosis. Sickle RBCs also undergo premature apoptosis which can lead to hemolytic anemia, which is a shortage of RBCs. Complications of SCD can include frequent infections, chronic inflammation, and vaso-occlusive crises (VOCs). Due to the hypercoagulable state of SCD, thrombotic complications can drive ischemic stroke, venous thromboembolism, severe pain episodes, and organ damage.

Coagulation factors are key instigators in the pathogenesis of SCD. Interestingly, the coagulation system can be activated and elevated in individuals with SCD, even in the absence of vascular occlusions. The coagulation pathway is initiated through two pathways, named the extrinsic pathway and the intrinsic pathway, that both operate through a final common pathway to initiate clot formation.

The extrinsic pathway of the coagulation cascade is normally activated through the release of tissue factor (TF) from endothelial cells following blood vessel injury. TF expression in vascular smooth muscle, pericytes, and fibroblasts of blood vessels is physically separated from its circulating ligand FVII/FVIIa by endothelium. However, in SCD the TF pathway is increased and abnormally expressed in circulating endothelial cells and monocytes. TF levels are additionally increased at locations of vascular damage where sub-endothelial TF is exposed to circulating blood. TF is constitutively produced by endothelial cells in SCD and increases of TF expression are often seen during VOC events. TF may also trigger inflammation and other pathways in addition to the coagulation cascade. This is supported by studies in animal models of SCD, where inhibition of TF reduces vascular inflammation and endothelial damage.

Components downstream of TF should also be considered when researching treatments for hypercoagulation and vascular injury in SCD. TF forms a complex with its ligand FVII following vascular injury. The activated TF-FVIIa complex in turn mediates the activation of FIX and FX. Activated FX, named FXa, forms a prothrombinase complex with FVa to convert prothrombin to thrombin. Thrombin functions to promote the cleavage of fibrinogen to fibrin. Clot formation occurs when fibrin fibers crosslink to form a meshwork that binds activated platelets. The extrinsic pathway activity can be measured as prothrombin time (PT).

The intrinsic pathway, also called the contact activation pathway, is activated by the exposure of endothelial collagen after damage to the vascular endothelium. The intrinsic pathway utilizes high-molecular-weight kininogen (HMWK), prekallikrein, and FXII to bind the vascular endothelial surface and activate FXIIa. FXIIa is a catalyst to initiate a cascade of factor activation involving FXIa, FIXa, and FVIIIa. The intrinsic pathway flows into a common pathway with the extrinsic pathway to activate FX.

Together, activated FX (FXa) and its cofactor FVa form a complex to cleave prothrombin to thrombin. Clot formation occurs following the activation of fibrin from fibrinogen. Thrombin activation also initiates a positive feedback loop to both the extrinsic and intrinsic pathways. The intrinsic pathway can be measured in activated partial thromboplastin time (aPTT). Interestingly, plasma levels of intrinsic pathway proteins, such as FXII, HMWK, and prekallikrein, are decreased in SCD individuals during steady states and during pain episodes.

In addition to the mentioned intrinsic pathway proteins, the intrinsic pathway can also be autoactivated on negatively charged surfaces, such as those containing phosphatidylserine (PS) or polyphosphates. This is mediated through the release of microparticles from apoptotic cells, RBCs, and platelets. Microparticles normally have procoagulant properties due to the presence of PS and TF on their membrane surfaces. In SCD, microparticles are primarily derived from RBCs and platelets and have elevated levels of negatively-charged PS on their membrane surfaces. This is important to note because PS-containing microparticles are able to bind intrinsic pathway proteins to promote thrombin generation. It is unclear if TF-positive microparticles are present in the blood of SCD individuals or if TF-positive microparticles contribute to coagulation activation during SCD. Therefore, it is suggested that microparticles promote coagulation in SCD through PS interactions with intrinsic pathway proteins in an autoactivation system.

Thrombin functions in the common coagulation pathway to prevent blood loss following vascular damage through the conversion of fibrinogen to fibrin and the activation of coagulation. Thrombin is an important mediator of inflammatory processes and can directly activate endothelial cells, platelets, and other cell types through PAR-1, PAR-3, and PAR-4 receptors. Thrombin also stimulates inflammatory proteins C5, protein C, and osteopontin. In SCD, thrombin levels are elevated as evidenced by increases in biomarkers of thrombin generation, such as thrombin-antithrombin complexes (TAT), prothrombin fragment 1.2 (F1.2), D-dimers, and plasmin-antiplasmin complexes (PAP). Ex vivo assays, such as thrombin generation assays (TGA), can also be used to analyze thrombin generation potential in plasma following a coagulation trigger.

During normal coagulation, thrombin cleaves soluble fibrinogen into insoluble fibrin. Fibrin is then crosslinked with platelets activated by FXIII to form a thrombus. An important step in the maintenance of coagulation is fibrinolysis. Fibrinolysis is the break down of crosslinked fibrin in blood clots. This process is mediated by plasmin. Degradation products, such as D-dimers, are removed by other proteases. Fibrinolysis activity is increased in SCD at steady state, and additionally elevated during VOC events. Biomarker analysis of fibrinolysis degradation products is useful for understanding the breakdown of clots during SCD. Fibrinolysis markers include urokinase-type plasminogen activator (uPA), Tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and D-dimers.

Additional Coagulation Factors

Platelets are essential to coagulation and hemostasis by promoting inflammation and thrombosis. Following blood vessel injury, platelets adhere to exposed collagen and Von Willebrand factor (vWF) in the subendothelium. Next, platelet activation occurs which enables platelets to aggregate and form a platelet plug. Fibrinogen bridges are necessary to stabilize activated platelets. Following fibrinogen cleavage, crosslinked fibrin forms a mesh that reinforces platelet plugs to complete the coagulation event.

In SCD, platelet numbers are elevated during steady state conditions and are further increased during VOC events. Functional assays suggest that platelet aggregation is elevated accompanied by an increase in platelet procoagulant activity in SCD, especially during acute pain events. Interestingly, activated platelets stimulate the adhesion of sickle RBCs to vascular endothelium during SCD, which promotes thrombosis. Increased platelet activation and aggregation can occur in SCD. Sickle hemoglobin, HbS, in the blood binds vWF in a mechanism that protects vWF from ADAMTS-13-mediated degradation. This enables vWF to accumulate in the blood. Further, multimeric vWF also enhances the adhesion of sickle RBCs to endothelium, compared to healthy RBCs. Together this makes vWF a powerful biomarker of the severity of endothelial damage during thrombosis in SCD.

The complement system is a member of the innate immune system that is also activated by the coagulation system. Hyperactivation of the complement system is associated with abnormal thrombosis. The complement system can be activated by sickle RBCs through exposure to PS or phosphatidylethanolamine in the alternative pathway. Degradation products that are released following RBC hemolysis can also stimulate the complement system. During SCD-associated VOC events, complement proteins C3a and C5a are elevated in the plasma and likely contribute to endothelial injury and vascular abnormalities in SCD.

Anticoagulant proteins naturally occur to manage and regulate coagulation and inflammation. Protein C and its cofactor protein S function to inhibit the coagulation factors FVa and FVIIIa. In SCD, depletion of protein C and protein S have been reported during steady state conditions and may contribute to the hypercoagulable characteristics of SCD. Clinical trials are underway to investigate the effectiveness of anticoagulant therapy to treat coagulopathies in SCD.

Summary

It is suggested that aberrant coagulation and platelet overactivation contribute to the pathogenesis of SCD. Further research and clinical trials are needed to explore the mechanisms driving abnormal coagulation in SCD and to identify effective therapies to treat it.

Coagulation Factors	Components	Function	Biomarkers and Methods of Evaluation	Activity in Sickle Cell Disease
Extrinsic Pathway	Tissue factor (TF), FVII	Primary pathway to initiate coagulation following vascular endothelial injury	Prothrombin time (PT)	Increased TF expression
Intrinsic Pathway	Prekallikrein HMWK, FXII, FXI	Activated by exposed collagen in the subendothelium and functions to initiate clot formation	Activated partial thromboplastin time (aPTT)	Decreased Prekallikrein, FXII, and HMWK plasma levels Increased levels of procoagulant microparticles
Common Pathway	FVIII, FIX, FX thrombin FXIII fibrin	Catalysts in the final steps of coagulation to cleave fibrinogen and form blood clots	Thrombin Generation Assays (TGA), TAT, PAP, F1.2, D-dimers, Fibrinogen	Increased thrombin levels
Fibrinolysis	Plasmin	Enzymatic degradation of fibrin in blood clots	uPA, tPA, PAI-1, D-dimers	Increased fibrinolytic activity
Platelet Activation	Collagen Fibrinogen, vWF ADAMTS-13	Platelets activate and aggregate to form a platelet plug	vWF, ADAMTS-13	Increased platelet numbers, platelet activation, and platelet aggregation
Complement System	C3a C5a	Activated by the coagulation system and contributes to thrombus formation	C3a and C5a	Increased C3a and C5a expression
Anticoagulant Proteins	Protein C Protein S	Negative feedback to regulate coagulation and inflammation	Protein C Protein S	Decreased Protein C and Protein S levels

Further Reading

Demagny, J. Driss, A. et al. ADAMTS13 and von Willebrand factor assessment in steady state and acute vaso-occlusive crisis of sickle cell disease. Res Pract Thromb Haemost. 2020 Dec 18;5(1):197-203. doi: 10.1002/rth2.12460.

Nasimuzzaman, Md and Malik, P. Role of the coagulation system in the pathogenesis of sickle cell disease. Blood Adv. 2019 Oct 22;3(20):3170-3180. doi: 10.1182/bloodadvances.2019000193.

Nouboussie, D. Key, NS and Ataga, K. Coagulation abnormalities of sickle cell disease: Relationship with clinical outcomes and the effect of disease modifying therapies. Blood Rev. 2016 Jul;30(4):245-56. doi: 10.1016/j.blre.2015.12.003.

Sparkenbaugh, E. and Pawlinski, R. Interplay between coagulation and vascular inflammation in sickle cell disease. Br J Haematol. 2013 Jul;162(1):3-14. doi: 10.1111/bjh.12336.

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