Thrombocytosis with acquired von Willebrand disease in an adolescent with sickle cell disease

Abstract One of the hallmark events of sickle cell disease (SCD) is vasoocclusion and episodic pain crisis. Although the mechanism of vascular occlusion is very complicated, processes like thrombosis and thromboembolism have been recognized to play an important role in the development of such clinical manifestation in SCD. Studies have shown that the von Willebrand factor (VWF), especially the ultra-large (UL) multimers play a major role in vasoocclusion, which clearly indicates a possible impairment of the VWF-cleaving metalloproteae ADAMTS-13 in these patients with SCD. In a recent work, indeed we have mentioned that the plasma ADAMTS-13 in patients with SCD having normal antigen level showed 35% less protease activity than the normal. There may be several plasma factors responsible for the acquired deficiency of ADAMTS-13 in SCD. Since, the increasing evidences suggest that the elevated level of extracellular hemoglobin (Hb) in plasma parallely associated with the pathogenesis of SCD, we investigated the effects of extracellular Hb on VWF proteolysis by ADAMTS-13. We observed that purified Hb dose-dependently inhibited the ADAMTS-13 cleavage of recombinant(r) VWF and endothelial ULVWF multimers under static and flow conditions. Hb bound to VWF multimers in a saturation-dependent manner and more potently to the rVWFA2 domain (affinity Kd~24nM), which contains the cleavage site for ADAMTS-13. Hb bound also to the ADAMTS-13 (Kd~65nM), with 2.7 times less affinity than to VWFA2. The bindings were neither calcium-dependent nor affected by haptoglobin. However, it is the Hb-binding to VWF that prevented the substrate from being cleaved by ADAMTS-13. These in vitro findings are consistent with the in vivo observations in patients with SCD. An elevated level of extracellular Hb in plasma was inversely correlated (linear regression, r2 =0.6354) with the low activity of ADAMTS-13 in a cohort of ten adult patients with SCD (mean±SE, Hb 346±138 mg/l; activity 33.3±30%) compared to age and gender-matched normal individuals (n=10; Hb 24±8 mg/l; activity 76.2±16%). The data together suggest that patients with SCD suffer from acquired ADAMTS-13 deficiency, primarily because Hb competitively binds and inhibits the proteolysis of VWF multimers, leading to ULVWF accumulation on vascular endothelium and in circulation. The Hb-VWF interaction may therefore be considered as a therapeutic target for reducing thrombotic and vasoocclusive complications in patients with severe hemolysis such as those with SCD.

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Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease

Blood ◽

10.1182/blood.2019003543 ◽

2020 ◽

Vol 135 (20) ◽

pp. 1783-1787 ◽

Cited By ~ 10

Author(s):

Erica M. Sparkenbaugh ◽

Chunsheng Chen ◽

Tomasz Brzoska ◽

Julia Nguyen ◽

Shaobin Wang ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Models ◽

Sickle Cell ◽

Factor Xa ◽

Organ Damage ◽

Cell Disease ◽

Thrombin Activation ◽

Protease Activated Receptor 1 ◽

Prophylactic Anticoagulation ◽

Von Willebrand

Abstract Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.

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Hemoglobin Bound Von Willebrand Factor (HbVWF) Multimers Are Hyperactive and More Prevalent in Patients with Sickle Cell Disease.

Blood ◽

10.1182/blood.v114.22.3058.3058 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3058-3058

Author(s):

Zhou Zhou ◽

Hyojeong Han ◽

Mark M. Udden ◽

Miguel A. Cruz ◽

Jing-fei Dong ◽

...

Keyword(s):

Cell Adhesion ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Von Willebrand Factor ◽

Percent Level ◽

Vascular Occlusion ◽

Cell Disease ◽

Normal Individuals ◽

Von Willebrand ◽

Willebrand Factor

Abstract Abstract 3058 Poster Board II-1034 Elevated levels of von Willebrand factor (VWF), especially the ultra-large multimers, play a significant role in the pathogenesis of vascular occlusion in sickle cell disease (SCD) by promoting cell adhesion to the endothelium. Investigating the pathophysiology of vaso-occlusion and thrombosis in SCD, we have recently observed that excessive extracellular-hemoglobin (Hb) in plasma significantly inhibited ADAMTS-13 proteolysis of VWF by binding directly to the enzyme cleavage-site on VWF. Here, we further show that subpopulations of VWF multimers, which are bound to extracellular-Hb, exist in plasma. We have successfully isolated the Hb-bound VWF (HbVWF) multimers from SCD patients' plasma using the Ni-NTA column and quantified by commercial kit. The HbVWF multimers exist in 5 to 6-times less quantity than the Hb-free multimers as measured in SCD patients. Purified HbVWF multimers are mostly uncleavable by recombinant ADAMTS-13 in vitro. These HbVWF multimers are hyper active in agglutinating platelets as detected by ristocetin cofactor (RCof) activity assay, and also hyper adhesive to collagen type-III compared to the Hb-free multimers. The HbVWF multimers exists in about 2-fold more quantity in SCD patients than normal individuals [mean percent level ± SE, 8.1±1.8 (individual mean 6 – 11) vs. 16.6±3 (12 – 21), P <0.001; n=10]. Using another sandwich-ELISA assay we have reexamined the HbVWF levels, which showed a similar pattern as above. Further, the increased level of HbVWF multimers exists parallely with an elevated RCof activity of plasma VWF [mean percent activity ± SE, 100.4±15.1 (78 – 124) vs. 132.9±11.4, (109 – 149), P <0.001] and high extracellular-Hb levels [mean mg/L ± SE, 59±6.5 (42 – 96) vs. 281.5±71.7 (184 – 410), P <0.001] in plasma of SCD patients compared to normal individuals. Therefore, we believe that these hyperactive HbVWF multimers play a crucial role in cell adhesion, vascular occlusion and thrombosis in SCD. Also, we speculate that this mechanism is not only limited in SCD, but also occurred in other pathophysiological conditions associated with severe intravascular hemolysis. Disclosures No relevant conflicts of interest to declare.

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Role of the coagulation system in the pathogenesis of sickle cell disease

Blood Advances ◽

10.1182/bloodadvances.2019000193 ◽

2019 ◽

Vol 3 (20) ◽

pp. 3170-3180 ◽

Cited By ~ 10

Author(s):

Md Nasimuzzaman ◽

Punam Malik

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Vascular Inflammation ◽

Coagulation Factor ◽

Organ Damage ◽

Coagulation System ◽

Cell Disease ◽

System Activation ◽

Vascular Occlusions ◽

Von Willebrand

AbstractSickle cell disease (SCD) is an inherited monogenic red blood cell disorder affecting millions worldwide. SCD causes vascular occlusions, chronic hemolytic anemia, and cumulative organ damage such as nephropathy, pulmonary hypertension, pathologic heart remodeling, and liver necrosis. Coagulation system activation, a conspicuous feature of SCD that causes chronic inflammation, is an important component of SCD pathophysiology. The key coagulation factor, thrombin (factor IIa [FIIa]), is both a central protease in hemostasis and thrombosis and a key modifier of inflammation. Pharmacologic or genetic reduction of circulating prothrombin in Berkeley sickle mice significantly improves survival, ameliorates vascular inflammation, and results in markedly reduced end-organ damage. Accordingly, factors both upstream and downstream of thrombin, such as the tissue factor–FX complex, fibrinogen, platelets, von Willebrand factor, FXII, high-molecular-weight kininogen, etc, also play important roles in SCD pathogenesis. In this review, we discuss the various aspects of coagulation system activation and their roles in the pathophysiology of SCD.

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ADAMTS13 and von Willebrand factor assessment in steady state and acute vaso‐occlusive crisis of sickle cell disease

Research and Practice in Thrombosis and Haemostasis ◽

10.1002/rth2.12460 ◽

2020 ◽

Author(s):

Julien Demagny ◽

Aurélie Driss ◽

Alain Stepanian ◽

Nadia Anguel ◽

Louis Affo ◽

...

Keyword(s):

Steady State ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Von Willebrand Factor ◽

Cell Disease ◽

Von Willebrand ◽

Willebrand Factor

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Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease

Frontiers in Immunology ◽

10.3389/fimmu.2020.613278 ◽

2021 ◽

Vol 11 ◽

Author(s):

Joan D. Beckman ◽

Fuad Abdullah ◽

Chunsheng Chen ◽

Rachel Kirchner ◽

Dormarie Rivera-Rodriguez ◽

...

Keyword(s):

Sickle Cell Disease ◽

Adhesion Molecules ◽

Sickle Cell ◽

Adhesion Molecule ◽

Tlr4 Expression ◽

Cell Disease ◽

Skin Fold ◽

Normal Human ◽

Complete Blood Counts ◽

Von Willebrand

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/-. These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.

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Haemoglobin blocks von Willebrand factor proteolysis by ADAMTS-13: A mechanism associated with sickle cell disease

Thrombosis and Haemostasis ◽

10.1160/th08-10-0677 ◽

2009 ◽

Vol 101 (06) ◽

pp. 1070-1077 ◽

Cited By ~ 63

Author(s):

Zhou Zhou ◽

Hyojeong Han ◽

Miguel Cruz ◽

José López ◽

Jing-Fei Dong ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Von Willebrand Factor ◽

Vascular Occlusion ◽

Dependent Manner ◽

Cell Disease ◽

Small Pool ◽

High Level ◽

Von Willebrand ◽

Willebrand Factor

SummaryVascular occlusion, thromboembolism and strokes are hallmark events in sickle cell disease (SCD). The von Willebrand factor (VWF), largest adhesive protein in circulation, has been implicated as major component in these processes. In SCD, a high level of extracellular haemoglobin (Hb) in plasma has been shown parallely associated with the disease pathogenesis. Investigating the effect of Hb we observed that purified Hb significantly inhibited the ADAMTS-13 cleavage of VWF under static and flow conditions. Hb bound potently to VWF specifically VWFA2 in a saturation-dependent manner with half-maximal binding 24 nM. Inversely, VWFA2 also bound potently to Hb and binding was inhibited by VP1 antibody, which binds to ADAMTS-13 cleavage site on VWF. Microscopic observation also shows that Hb bound specifically to endothelial VWF under flow. Furthermore, the Hb-bound VWF multimers were isolated from plasma. Though, Hb bound also to ADAMTS-13, it is the Hb binding to VWFA2 that prevented the substrate being cleaved by ADAMTS-13. In an observation in a small pool of patients with SCD, high Hb in plasma was inversely correlated with low proteolytic activity of ADAMTS-13. Thus, the observations suggest that the patients with SCD suffer from an acquired ADAMTS-13 deficiency primarily because Hb competitively bound and blocked the proteolysis of VWF, leading to the accumulation of ultra-large VWF multimers in circulation and on endothelium. Therefore, the Hb-VWF interaction may be considered as a therapeutic target for treating thrombotic and vaso-occlusive complications in patients with severe intravascular haemolysis such as those with SCD.

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