Shuju Feng

Partial ADAMTS13 deficiency in atypical hemolytic uremic syndrome

Complement dysregulation leads to atypical hemolytic uremic syndrome (aHUS), while ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura. We investigated whether genetic variations in the ADAMTS13 gene partially explain the reduced activity known to occur in some patients with aHUS. We measured complement activity and ADAMTS13 function, and completed mutation screening of multiple complement genes and ADAMTS13 in a large cohort of aHUS patients. In over 50% of patients we identified complement gene mutations. Surprisingly, 80% of patients also carried at least 1 nonsynonymous change in ADAMTS13, and in 38% of patients, multiple ADAMTS13 variations were found. Six of the 9 amino acid substitutions in ADAMTS13 were common single nucleotide polymorphisms; however, 3 variants-A747V, V832M, and R1096H- were rare, with minor allele frequencies of 0.0094%, 0.5%, and 0.32%, respectively. Reduced complement and ADAMTS13 activity (<60% of normal activity) were found in over 60% and 50% of patients, respectively. We concluded that partial ADAMTS13 deficiency is a common finding in aHUS patients and that genetic screening and functional tests of ADAMTS13 should be considered in these patients.

Filamin A Binding Stabilizes Nascent Glycoprotein Ibα Trafficking and Thereby Enhances Its Surface Expression

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIbα possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbα directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbα biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbα with GpIbβ, GpIX with and without filamin A. We observed that surface GpIbα expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbα, GpIbβ, and GpIX (CHO-GpIbα/βIX), lysates from CHO-GpIbα/βIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbα, but lesser amounts of the ∼15-kDa C-terminal peptide released when the extracellular domain of GpIbα is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbα at C-terminal residue 557 or by a deletion between amino acids 560–570, the decreased synthesis of mature GpIbα is accompanied by decreased immature GpIbα and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIbα in CHO-GpIbα/βIX cells is eliminated by brefeldin A (which inhibits transport out of the endoplasmic reticulum (ER)) and restored by lactacystin (which inhibits proteasomal degradation). These results suggest that GpIbα binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIbα to the cell surface.

Von Willebrand Factor is a cofactor in complement regulation

Several complement proteins interact with hemostatic factors. We discovered that von Willebrand factor (VWF) acts as a cofactor for factor I-mediated cleavage of complement C3b, thereby shutting down complement activation. The complement regulatory function of VWF multimers depends on their size. Smaller VWF multimers enhance cleavage of C3b but large and ultra-large VWF (ULVWF) multimers have no effect on C3b cleavage and permit default complement activation. We conclude that normal plasma VWF multimers prevent complement activation and steer the complement pathway toward generation of inactivated C3b (iC3b). ULVWF multimers, as are present in patients with thrombotic microangiopathy, lack an inhibitory effect on complement and permit complement activation.

Human Complement Factor H Is a Reductase for Large Soluble von Willebrand Factor Multimers—Brief Report

Objective—Ultralarge von Willebrand factor (vWF) strings are secreted by, and anchored to, stimulated human endothelial cells. A disintegrin and metalloprotease with thrombospondin domains-type 13 cleaves the ultralarge vWF strings into large soluble vWF multimers. Normal plasma contains a nonproteolytic reducing activity that subsequently rapidly diminishes the size of the large soluble vWF multimers. Approach and Results—The vWF reductase activity was isolated from normal cryoprecipitate-poor plasma by chromatography and identified as the complement regulatory protein, factor H (FH), by mass spectroscopy, SDS-PAGE, and monospecific anti-FH antibody. Removal of FH from partially purified vWF reductase by immunoabsorption eliminated the reducing activity, and the activity was recovered in the eluates. Recombinant human FH reduced large soluble vWF multimers in a free thiol-dependent reaction that was not inhibited by a variety of protease inhibitors. Conclusions—FH contributes to the reduction of large soluble vWF multimers.

The Interaction between Factor H and Von Willebrand Factor

Complement factor H (fH) is a plasma protein that regulates activation of the alternative pathway, and mutations in fH are associated with a rare form of thrombotic microangiopathy (TMA), known as atypical hemolytic uremic syndrome (aHUS). A more common TMA is thrombotic thrombocytopenic purpura, which is caused by the lack of normal ADAMTS-13-mediated cleavage of von Willebrand factor (VWF). We investigated whether fH interacts with VWF and affects cleavage of VWF. We found that factor H binds to VWF in plasma, to plasma-purified VWF, and to recombinant A1 and A2 domains of VWF as detected by co-immunoprecipitation (co-IP) and surface plasmon resonance assays. Factor H enhanced ADAMTS-13-mediated cleavage of recombinant VWF-A2 as determined by quantifying the cleavage products using Western-blotting, enhanced cleavage of a commercially available fragment of VWF-A2 (FRETS-VWF73) as determined by fluorometric assay, and enhanced cleavage of ultralarge (UL) VWF under flow conditions as determined by cleavage of VWF-platelet strings attached to histamine stimulated endothelial cells. Using recombinant full-length and truncated fH molecules, we found that the presence of the C-terminal half of fH molecule is important for binding to VWF-A2 and for enhancing cleavage of the A2 domain by ADAMTS-13. We conclude that factor H binds to VWF and may modulate cleavage of VWF by ADAMTS-13.

Glycoprotein Ib/IX/V binding to the membrane skeleton maintains shear-induced platelet aggregation.

The extracellular domain of glycoprotein (Gp) Ibalpha serves as the von Willebrand factor (vWf) receptor that triggers shear stress-dependent platelet aggregation. Its intracellular domain associates with actin-binding protein-280 (filamin 1a) that binds directly to filamentous actin, thereby linking the membrane skeleton to GpIbalpha. We examined the functional significance of GpIbalpha interactions with actin during platelet aggregation in response to 120 dyn/cm(2) shear stress. Lysates of resting and sheared platelets were centrifuged at approximately 13,000xg for 15 min, and GpIbalpha was immunoprecipitated from the lysate supernatant. GpIbalpha and coimmunoprecipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies specific for GpIbalpha and actin. We observed a significant increase in the amounts of actin coimmunoprecipitating with GpIbalpha as platelets aggregated in response to shear stress. Actin/GpIbalpha interactions reached a maximum after 90 s of shear stress. Monoclonal antibody (mAb) blockade of vWf binding to GpIbalpha inhibited shear stress-induced platelet aggregation and actin associating with GpIbalpha. Pretreatment of platelets with cytochalasin D resulted in the inhibition of actin binding to GpIbalpha in sheared platelets and in an increase in the rate and magnitude of platelet disaggregation. These data indicate that shear stress causes changes in the association between GpIbalpha and the actin-based membrane skeleton. The increased interaction between GpIbalpha and the actin-based membrane skeleton results from shear-induced vWf binding to GpIbalpha and is mechanoprotective in that it maintains shear-induced aggregation of activated platelets.

Complement activation in thrombotic microangiopathies.

The thrombotic microangiopathies (TMA) are a group of disorders defined by the presence of microangiopathic hemolytic anemia and thrombocytopenia. The most common of these is thrombotic thrombocytopenic purpura (TTP), which is a systemic disorder of microvascular thromboses due to deficiency of ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). A less common TMA is the atypical hemolytic uremic syndrome (aHUS), which is a renal vascular TMA caused by complement dysregulation. Despite overlapping clinical and pathologic manifestations, TTP and aHUS have distinct etiologies. TTP is often caused by a deficiency of ADAMTS-13 that is the result of gene mutations or acquired autoantibodies (Tsai, 2006). Atypical HUS is caused by defects of regulation and/or excessive activation of the alternative complement pathway (Kavanagh & Goodship, 2010). The mechanism by which complement dysregulation contributes to aHUS is not precisely defined, although complement-mediated glomerular endothelial injury and enhanced complement-mediated platelet activation are probably involved (Stahl et al, 2008). Similarly, triggers and co-factors directing systemic platelet deposition in TTP are not completely understood. Evidence that complement activation might play a role in TTP (Noris et al, 1999; Ruiz-Torres et al, 2005; Reti et al, 2012) raises the possibility of a cross-talk between ADAMTS-13/ultra-large von Willebrand factor (ULVWF) and the complement system. We studied plasma samples of 81 patients diagnosed with TMA according to clinical criteria for functional abnormalities in both ADAMTS-13 and complement regulation. Citrated platelet-poor plasma samples were obtained for testing before the initial plasma infusion or exchange procedures. All patients had microangiopathic hemolytic anemia and thrombocytopenia without an alternative cause, and treated with either plasma infusion or plasma exchanges. None of our patients had acute renal failure. Samples for analysis of DNA were not obtained/stored from this group of patients. All human subject studies were conducted according to the approved institutional review board protocols in the Rice University and University of Texas M.D. Anderson Cancer Center. ADAMTS-13 activity was measured by: (1) the rate of cleavage of a substrate that contains 73 amino acids of the A2 domain of VWF with fluorescence resonance energy transfer (FRET) tags on either side of the cleavage site for ADAMTS-13 (FRETS-VWF73), according to the manufacturer’s protocol (GTi Diagnostics); and (2) cleavage of urea-treated ULVWF multimers (obtained from human umbilical vein endothelial cell supernatant) by citrated patient plasma, followed by VWF multimeric analysis using SDS-1% agarose electrophoresis and Western-blotting with anti-VWF antibody. This is a modification of the method described by Furlan, et al. (Furlan et al, 1998). The presence or absence of ADAMTS-13 inhibitors was determined by measuring cleavage of urea-treated ULVWF multimers before and after mixing normal citrated plasma with an equal volume of patient citrated plasma (Furlan et al, 1998). Complement activity was measured by the hemolysis of sheep erythrocytes after incubation with human serum or plasma according to modified techniques from Sanchez-Corral, et al. (Sanchez-Corral et al, 2004). Factor H-depleted plasma causes complement-induced lysis of sheep erythrocytes with the visually apparent release of hemoglobin. Pooled normal plasma or serum caused 7% and 8% hemolysis of sheep erythrocytes, respectively. Optimal dilution of plasma or serum for the assay was determined to be between 4/100 to 6/100, and optimal incubation time was 10 min. Sixty percent (49/81) of TMA patients had severe ADAMTS-13 deficiency (less than 10% activity). Eighty percent (65/81) of our patients’ plasma samples caused little to no hemolysis of sheep erythrocytes (median of 10%; range of 0–15%). In contrast, 20% (16/81) of the patients’ samples showed significant hemolysis (median of 60% hemolysis; range of 23–89%) (Figure 1). Sixteen percent (8/49) of plasma samples from TTP patients with severe ADAMTS-13 deficiency caused increased hemolysis. Only one of the 8 patients with concurrent excessive complement-induced hemolysis and severe ADAMTS-13 deficiency had detectable antibody (in low titer) against ADAMTS-13 (Table 1). Twenty-five percent (8/32) of plasma samples from patients who did not have severe ADAMTS-13 deficiency also caused increased hemolysis. Figure 1 Sheep erythrocyte hemolysis assay in plasma samples from patients with thrombotic microangiopathy Table 1 Patients with a clinical diagnosis of TTP and decreased complement regulation. Severe deficiency of functional ADAMTS-13 is associated with TTP; however, many patients with a TTP-like syndrome have normal ADAMTS-13 levels, as did 40% (32/81) of the patients in our study. There are several reports of patients with reduced ADAMTS-13 function who either did not develop TTP, or did so later in life (Noris et al, 2005). These observations raise the possibility of the presence of additional factors besides ADAMTS-13 deficiency involved in the pathophysiology of TTP (Ruiz-Torres et al, 2005; Reti et al, 2012; Noris et al, 2005; Chapin et al, 2012). Activation of the complement system in both familial (Noris et al, 1999) and acquired TTP (Reti et al, 2012) has been reported, based on the lower concentration of C3 and elevated levels of complement activation products (C3a and sC5b-9) in the sera of patients with acute TTP, and deposition of C3 and C5b-9 on endothelial cell exposed to TTP sera (Ruiz-Torres et al, 2005). We studied activity of the alternative complement pathway in 81 patients with the clinical diagnosis of TTP-like TMA requiring plasma infusion and/or plasma exchange. Some patients with severe ADAMTS-13 deficiency (8/49; 16%) or TTP-like TMA (8/32; 25%), had elevated plasma complement activity. We did not detect an increased titer of ADAMTS-13 inhibitor in ADAMTS-13 deficient TTP patients with complement dysregulation, and the majority of these patients (5/8; 68%) had a history of familial or recurrent TTP (Table 1). Our data suggest that the complement system may be an important co-factor involved in the pathogenesis of TMA. Excessive alternative pathway activity occurred in a significant number of TTP patients indicating that concurrent defects in ADAMTS-13 and complement regulation may occur more frequently than previously reported (Noris et al, 2005; Chapin et al, 2012). In addition, our findings indicate that excessive alternative pathway activity can be associated with a TTP-like TMA in some patients who do not have severe deficiencies of ADAMTS-13. Further genetic studies of patients with the clinical diagnosis of TTP may be informative.

Targeting shear stress-induced platelet activation: is lesion-specific antiplatelet therapy a realistic clinical goal?

Platelets are mediators of physiologic hemostasis and pathologic thrombosis. They operate within distinctive vascular and rheologic microenvironments, and their participation in hemostasis or thrombosis is directed by distinct variables operating within the microenvironment. Thrombosis is not simply too much hemostasis: there is good evidence that triggering mechanisms of platelet aggregation under low shear stress conditions are different from those operating under high shear stress conditions. Such differences are hypothesized to exist in vivo and to separate mechanisms of microvascular hemostasis from mechanisms of arterial thrombosis, such as those involved in myocardial, cerebral and peripheral vascular ischemia and infarction. This separation forms the conceptual basis for the hypothesis that lesion-specific antithrombotic agents might some day be invented that inhibit arterial thrombosis without causing bleeding that arises from impaired hemostasis. The focus of much of the work in this field has been platelet aggregation initiated by shear-dependent von Willebrand factor binding to the platelet glycoprotein Ib-IX-V complex. It is hypothesized that by elucidating molecular mechanisms of platelet activation operating under pathologically elevated shear stresses, targets of lesion-specific therapies will one day be identified for use in clinical syndromes of arterial thrombosis.

Platelets are not hyperreactive in patients with ovarian cancer

Abstract Paraneoplastic thrombocytosis has been reported in different types of solid tumors, including ovarian epithelial cancer, and found to be associated with a worse outcome. Although the effect of cancer on increasing platelet counts is well documented, the effect of cancer on platelet functions is not well known. We compared in vitro aggregation response of platelets isolated from 34 patients with ovarian cancer to those of platelets from 19 patients with benign ovarian tumors. Aggregation studies were conducted in a light transmission aggregometer, using both a high and a low dose of ADP and collagen. We evaluated platelet preactivation by measuring the plasma concentration of β-thromboglobulin (β-TG) and platelet factor-4 (PF-4) as markers of platelet α granule secretion, using ELISA. We found that ovarian cancer is not associated with an enhanced aggregation response of platelets to ADP or collagen, and plasma concentration of β-TG and PF-4 is not higher in patients with ovarian cancer compared to those in patients with benign ovarian tumors.