Role of A and B blood group antigens in the expression of adhesive activity of von Willebrand factor.

ABO (H) blood group antigens are covalently linked to the oligosaccharide side-chains of von Willebrand factor (VWF). In this study, we investigated the role of the A and B antigens in the expression of VWF adhesive activity. VWF of type A, B or O was purified from fresh frozen plasma. Presence of A or B antigen on the VWF was confirmed by enzyme-linked immunosorbent assay (ELISA) and by immunoblotting with monoclonal anti-A or anti-B. The A or B antigen was also detected in the 48/52-kDa fragment of the respective VWF after trypsin digestion. Removal of A antigen with alpha-N-acetylgalactosaminidase or B antigen with alpha-galactosidase did not affect its multimer size or antigenic level, but decreased the ristocetin cofactor (RCoF) activity of the respective VWF by 33-39% (P < 0.01-0.002). Removal of A or B antigen from VWF did not affect the binding of the VWF to immobilized type III collagen. A and B antigens were not detected in platelet VWF. These results indicate that AB structures play a role in platelet aggregating activity of VWF.

Proteolytic cleavage of recombinant type 2A von Willebrand factor mutants R834W and R834Q: inhibition by doxycycline and by monoclonal antibody VP-1.

The susceptibility of recombinant type 2A von Willebrand factor (vWF) to a recently identified plasma metalloproteinase and the potential application of proteolysis inhibition in the treatment of the disease were investigated. Two recombinant type 2A vWF mutants, R834W and R834Q, were spontaneously cleaved by the partially purified plasma proteinase to smaller forms. When treated with guanidine HCI, both the wild-type and the R834W mutant vWF exhibited a biphasic change in proteolytic susceptibility, reaching the same maximum cleavage at 1.25 mol/L guanidine HCI. Proteolysis of the recombinant vWF generated the same 350-kD and 200-kD species (dimers of the 176-kD and 140-kD fragments, respectively) as those found in normal plasma. The proteinase activity was inhibited by doxycycline, with an IC50 of approximately 0.25 mmol/L. The inhibitory activity of doxycycline was related to its metallic cation binding activity. Susceptibility of the recombinant vWF to the proteinase was inhibited by monoclonal antibody VP-1 (directed against residues 828-842 of the vWF polypeptide), but not by two other monoclonal antibodies M13 and M31. The spontaneous susceptibility to proteolytic cleavage may account for the lack of large multimers in type 2A von Willebrand disease (vWD), and the results with tetracyclines and monoclonal antibody VP-1 offer new strategies for developing specific treatment of type 2A vWD.

Platelet alpha granule deficiency associated with decreased P-selectin and selective impairment of thrombin-induced activation in a new patient with gray platelet syndrome (alpha-storage pool deficiency).

We report studies on a new patient with gray platelet syndrome (GPS, alpha-storage pool deficiency). Her lifelong bleeding history is associated with platelet abnormalities characteristic of GPS including mild to moderate thrombocytopenia, a population of abnormally large platelets, and specific deficiencies of alpha-granule constituents and morphologically typical alpha-granules. Platelet function studies showed normal aggregation responses to adenosine diphosphate, epinephrine, collagen, arachidonate, and ristocetin but impaired activation responses to thrombin and a thrombin receptor-activating peptide (T1 peptide). These impaired responses included T1 peptide-induced aggregation, thrombin-induced adenine nucleotide secretion, and thrombin-induced (Ca2+)i increases. The impairment of the thrombin-induced (Ca2+)i increase was observed as a substantially slower initial rise in (Ca2+)i levels and a smaller maximum (Ca2+)i increase compared with the responses obtained in normal platelets and are thus similar to those reported previously in another patients with GPS. Flow cytometric measurements of the binding of two distinct monoclonal antibodies against the functional thrombin receptor indicated the presence of a normal number of receptors and normal receptor cleavage by thrombin in the GPS platelets, providing additional support for the hypothesis presented in previous studies that the thrombin activation defect in GPS platelets occurs subsequent to the interaction of thrombin with its receptor. The alpha-granule deficiency in this patient was associated with an approximately 50% decrease in the content and surface expression of the alpha-granule membrane-specific protein P-selectin in contrast to a previous report of normal amounts of P-selectin in the platelets of two related patients with GPS. This finding raises the possibility that the alpha-granule deficiency in GPS may be expressed in different phenotypes characterized by differences in the amount or constitution of residual alpha-granule membranes present in GPS platelets.

A distinct coagulopathy associated with interleukin-2 therapy.

We studied the coagulation profiles of 14 patients with advanced malignancies treated with Interleukin-2 (IL-2). A 43% prolongation of the PTT (P < 0.001) and a significant decrease in functional levels of factors II, IX, X, XI, and XII were observed 6 h post IL-2 treatment in comparison to pretreatment values. These parameters normalized within 2-3 d following IL-2 administration. The PT, factors V, VII, VIII, fibrinogen and D-dimer levels were unchanged with IL-2 treatment. This pattern of coagulopathy has not previously been reported.

Shear stress enhances the proteolysis of von Willebrand factor in normal plasma.

While von Willebrand factor (vWF) is secreted from endothelial cells as a very large polymer, it circulates as a series of multimers that are reducible to a 225-kD polypeptide and three proteolytic fragments of 189, 176, and 140 kD. Cleavage at the Tyr-842/Met-843 bond of the vWF polypeptide creates the 140- and 176-kD fragments. In the process of understanding vWF multimer formation, the role of shear stress in vWF proteolysis was investigated in this study. A shear-rate-dependent loss of the largest multimers was observed when normal plasma was perfused through long capillary tubings achieving shear rates normally encountered in the circulation. The shear-dependent vWF change was not observed when purified vWF or normal plasma containing calcium chelator EGTA or EDTA was perfused. As the large multimers decreased, an increase in the smaller multimers, including 200- and 350-kD bands, was detected. Elution and immunoblotting studies with peptide-specific antibodies LJ-7745 and VP-1 showed that the 200-kD band was a dimer of the 140-kD fragment, whereas the 350-kD band was a dimer of the 176-kD fragment. When analyzed after disulfide bonds were reduced, sheared plasma showed an increase in the 176- and 140-kD fragments, but not the 189-kD fragment. Finally, shearing of purified vWF enhanced its proteolytic cleavage when it was subsequently incubated with the cryosupernatant fraction of normal plasma or with cathepsin G, a leukocyte granule serine protease. These results show that shear stress is capable of enhancing the susceptibility of vWF to proteolytic cleavage. It promotes vWF proteolysis in normal plasma at a site that generates the 140-kD/176-kD fragments, leading to a decrease in multimer size. Shear stress might be involved in modulating the size of vWF in the circulation.

Differential effect of metabolic fuels on the energy state and Na(+)-K(+)-ATPase in isolated cerebral microvessels.

Isolated bovine cerebral microvessels (ICMV) were incubated with different metabolic fuels to determine their ability to support microvessel Na(+)-K(+)-ATPase (quantitated as ouabain-sensitive 86Rb+ uptake) and the ATP/ADP ratio. In comparison with ICMV incubated with glucose, Na(+)-K(+)-ATPase activity was reduced by 55% after a 3-h incubation in fuel-free medium and by 30-40% after incubation with beta-hydroxybutyrate, acetoacetate, or glutamate. However, Na(+)-K(+)-ATPase activity was not significantly decreased in ICMV incubated with pyruvate or oleate plus carnitine. In contrast, only glucose was able to maintain the ATP/ADP ratio. To evaluate the effect of endogenous fatty acid metabolism on these parameters, ICMV were incubated with bromostearate, an inhibitor of fatty acid oxidation. Bromostearate decreased both Na(+)-K(+)-ATPase activity and the ATP/ADP ratio, even in the presence of glucose. These results indicate that the varying effects of different fuels on Na(+)-K(+)-ATPase in ICMV cannot be explained solely by their effects on the ATP/ADP ratio or on glycolytic ATP generation. They suggest that other fuel-modulated factors play a key role in regulating this enzyme.

Endothelium-dependent inhibition of Na(+)-K+ ATPase activity in rabbit aorta by hyperglycemia. Possible role of endothelium-derived nitric oxide.

Hyperglycemia has been shown to diminish Na(+)-K+ ATPase activity in rabbit aorta. To examine the basis for this effect, aortic rings were incubated for 3 h in Krebs-Henseleit solution containing 5.5 or 44 mM glucose, and Na(+)-K+ ATPase activity was then quantified on the basis of ouabain-sensitive (OS) 86Rb-uptake. Incubation with 44 mM glucose medium caused a 60% decrease in Na(+)-K+ ATPase activity in rings with intact endothelium (from 0.22 +/- 0.01 to 0.091 +/- 0.006 nmol/min per mg dry wt; P less than 0.01). Similar decreases (45%; P less than 0.01) in Na(+)-K+ ATPase activity were seen when rings incubated with 5.5 mM glucose were exposed to NG-monomethyl L-arginine (300 microM), an inhibitor of endothelium-derived nitric oxide (EDNO) synthesis or when the endothelium was removed (43% decrease). The decrease in Na(+)-K+ ATPase activity induced by hyperglycemia was totally reversed upon adding to the medium either L-arginine, a precursor of EDNO biosynthesis or sodium nitroprusside, which bypasses endothelium and directly activates the soluble guanylate cyclase in vascular smooth muscle. A decrease in Na(+)-K+ ATPase activity (42%; P less than 0.05), only seen in the presence of endothelium, was also observed in aortas taken directly from alloxan-induced diabetic rabbits. These studies suggest that the decrease in vascular Na(+)-K+ ATPase activity induced by hyperglycemia is related, at least in part, to a decrease in the basal release of EDNO. They also suggest that alterations in basal EDNO release and possibly Na(+)-K+ ATPase activity contribute to the impairment in vascular relaxation caused by hyperglycemia and diabetes.

The high molecular weight form of endothelial cell von Willebrand factor is released by the regulated pathway.

We have previously reported that two forms of von Willebrand factor (vWf) exist in cultured human umbilical vein endothelial cells: a high molecular weight (HMW) form that is released and can be proteolytically cleaved into a series of plasma-like multimers, and a non-secreted low molecular weight (LMW) form. In this study, the mode of vWf release and the relationship between the two forms were examined. As determined by two-dimensional analysis as well as by immunoreactivity with an antibody to the propolypeptide, the LMW form of endothelial cell vWf consisted of a 260 kD pro-vWf polypeptide, while the HMW form consisted of a 225 kD mature polypeptide. Only the 260 kD polypeptide was susceptible to digestion with endoglycosidase H. Release of the HMW form into the culture media was accompanied by a decrease in cellular vWf. Treatment of endothelial cells with cycloheximide or tunicamycin caused a decrease in the LMW form but did not affect the secretion of the HMW form. These results suggest that two pools of vWf exist in endothelial cells--a LMW form of pro-vWf in the endoplasmic reticulum and a HMW form of mature vWf in the storage compartment. Released vWf derives only from the storage pool.

Subunit composition of plasma von Willebrand factor multimers: evidence for a non-proteolytic mechanism resulting in apparent increase in proteolytic fragments.

Plasma von Willebrand factor (vWf) consists of a series of multimers of different molecular sizes. When analyzed for subunit composition, plasma vWf contained a major polypeptide of approximately 225 kD, and at least three smaller proteolytic fragments. In the present study, the subunit composition of each of these multimers was analyzed using a two dimensional electrophoresis technique. Although nearly all the multimers, as separated by SDS 1.5% agarose gel electrophoresis, contained both the major polypeptide and the smaller fragments, the relative amount of the fragments varied among the multimers. The smaller multimers contained relatively more proteolytic fragments. After the larger multimers were depleted by incubation of plasma with formaldehyde-fixed platelets and ristocetin, the intact subunit decreased, while the amount of the proteolytic fragments remained relatively unchanged. The findings of this study are consistent with the scheme that plasma multimers undergo proteolytic cleavage after multimer assembly. Furthermore, an apparent increase in the amount of these proteolytic fragments may result from mechanisms unrelated to proteolysis, such as selective adsorption of the larger multimers onto platelets.

Endothelin stimulates Na(+)-K(+)-ATPase activity by a protein kinase C-dependent pathway in rabbit aorta.

Incubation with endothelin (Endo) caused a time- and concentration-dependent increase in both ouabain-sensitive (OS) and ouabain-insensitive (OI) 86Rb+ uptake [half-maximal effective concentration (EC50) for OS component = 11 nM] in the rabbit aorta. Increase in the OS component [Na(+)-K(+)-adenosine triphosphatase (ATPase) activity] accounted for 70% of the 110% increase in total 86Rb+ uptake at a maximally effective concentration of Endo (100 nM). Protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBU; 100 nM) increased total 86Rb+ uptake by 69%, with 42% of the increase in the OS component. Stimulation by Endo and PDBU was not additive. Staurosporine (STA; 100 nM) inhibited stimulation of total 86Rb+ uptake by Endo and PDBU by approximately 60%. With ouabain and STA added together, inhibition of Endo-stimulated total 86Rb+ uptake (90%) was greater than with either agent alone, suggesting that STA inhibits an OS as well as an OI component of 86Rb+ uptake. Stimulation of total 86Rb+ uptake by both Endo and PDBU were also inhibited by approximately 60% by the Na(+)-H+ exchange inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Endo-stimulated total 86Rb+ uptake was not further inhibited when ouabain was added together with EIPA, suggesting that Na(+)-H+ exchange is primarily linked to the OS component of 86Rb+ uptake. In contrast, Na(+)-K(+)-Cl- cotransport inhibitor bumetanide inhibited increases in total 86Rb+ uptake caused by Endo (30%) and PDBU (56%) due solely to its effects on OI 86Rb+ uptake. Results suggest that Endo stimulates Na(+)-K(+)-ATPase activity in rabbit aorta by activating PKC and Na(+)-H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of Plasmodium falciparum in human erythrocytes containing abnormal membrane proteins.

To evaluate the role of erythrocyte (RBC) membrane proteins in the invasion and maturation of Plasmodium falciparum, we have studied, in culture, abnormal RBCs containing quantitative or qualitative membrane protein defects. These defects included hereditary spherocytosis (HS) due to decreases in the content of spectrin [HS(Sp+)], hereditary elliptocytosis (HE) due to protein 4.1 deficiency [HE(4.1(0))], HE due to a spectrin alpha I domain structural variant that results in increased content of spectrin dimers [HE(Sp alpha I/65)], and band 3 structural variants. Parasite invasion, measured by the initial uptake of [3H]hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp+) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. Preincubation of HS(Sp+) RBCs in culture for 3 days did not alter these results. Normal parasite growth was observed in RBCs from one HS subject with normal membrane spectrin content. The extent of decreased parasite growth in HS(Sp+) RBCs closely correlated with the extent of RBC spectrin deficiency (r = 0.90). Homogeneous subpopulations of dense HS RBCs exhibited decreased parasite growth to the same extent as did HS whole blood. RBCs from four HE subjects showed marked parasite growth inhibition, the extent of which correlated with the content of spectrin dimers (r = 0.94). RBCs from two unrelated subjects with structural variants of band 3 sustained normal parasite growth. Decreased growth in the pathologic RBCs was not the result of decreased ATP or glutathione levels or of increased RBC hemolysis. We conclude that abnormal parasite growth in these RBCs is not the consequence of metabolic or secondary defects. Instead, we suggest that a functionally and structurally normal host membrane is indispensable for parasite growth and development.

Inhibition of von Willebrand factor-induced platelet agglutination by ADP does not result from reduced binding of total von Willebrand factor or its larger multimers.

Earlier experiments showed that platelet agglutination induced by von Willebrand factor (vWf) plus ristocetin was greatly diminished if adenosine diphosphate (ADP) was added first in the presence of ethylenediaminetetraacetic acid (to prevent aggregation). Platelets treated with ADP and then fixed also agglutinated less than control fixed platelets. The studies reported here demonstrate that ADP did not decrease ristocetin-induced binding of vWf whether binding was measured on suspended platelets with iodine 125-labeled vWf or on suspended or agglutinated platelets with the use of any of three 125I-labeled monoclonal antibodies that bind to vWf but that do not interfere with ristocetin-induced agglutination. Equal amounts of vWf were eluted from ristocetin/vWf-treated platelets when they were resuspended without ristocetin, whether or not the platelets had been exposed to ADP, and the vWf recovered in either case was composed only of large multimers. No evidence for an agglutination site other than glycoprotein Ib could be demonstrated by measuring agglutination of a mixture of platelets fixed after inhibition with antibody against glycoprotein Ib and platelets fixed after inhibition with ADP. We conclude that inhibition of agglutination by ADP must involve the way in which vWf is bound, because it does not result from a decreased amount or from a difference in multimer size of bound vWf.

Glucose is required to maintain ATP/ADP ratio of isolated bovine cerebral microvessels.

Isolated bovine cerebral microvessels (ICMV) were incubated with different metabolic fuels to determine the effect of each of them on microvessel energy state. With no fuel added to the medium, the ATP/ADP generally decreased from initial values of 1.5-3 down to 1-1.5 over 4 h; the ATP content also declined approximately 50%. In contrast, with glucose present, the ATP/ADP increased, and the ATP content was maintained. Pyruvate, beta-hydroxybutyrate, glutamate, and oleate were ineffective; oleate added together with carnitine gave some improvement but less than with glucose. Oxygen consumption by ICMV did not differ appreciably in fuel-free or glucose-containing medium. Addition of an inhibitor of fatty acid oxidation, 2-tetradecylglycidate, depressed the ATP/ADP. These results suggest that ICMV require glycolysis to maintain both their content of ATP and their ATP/ADP. They also suggest that endogenous lipid is an important fuel for isolated microvessels.

Postoperative thrombocytopenia in type IIB von Willebrand disease.

We report studies of a large kindred with type IIb von Willebrand disease and manifestations of thrombocytopenia. While only one member of the family was thrombocytopenic routinely, three members of the family who underwent various surgical procedures demonstrated thrombocytopenia and platelet clumping postoperatively. Platelet clumps were found on peripheral blood smear only in the immediate postoperative specimens and did not appear to be a technical artifact. In the one patient who received no preoperative prophylactic therapy, postoperative plasma specimens showed the transient appearance of high molecular weight von Willebrand factor multimers. These results support the hypothesis that surgery, or some related aspect such as stress, led to the release of high molecular weight multimers, resulting in platelet clumping and removal from the circulation, and subsequent thrombocytopenia. Thrombocytopenia under conditions of stress may be a more common manifestation of type IIb vWd than is currently appreciated.

Desmopressin induces adhesion of normal human erythrocytes to the endothelial surface of a perfused microvascular preparation.

The interaction of red blood cells (RBCs) with vascular endothelium under flow conditions was investigated using the perfused rat mesocecum. Under videomicroscopy, normal human erythrocytes were found to adhere to the venular endothelium of desmopressin-treated microvasculature. Transmission electron microscopy showed that the erythrocytes were attached to the endothelial cells at discrete electron-dense sites. Compared with control preparations in which the microvasculature was perfused with Ringer's-albumin solution alone, more than a 10-fold increase in radioactivity was retained in the desmopressin-treated microvasculature when technetium (99mTc)-labeled erythrocytes were infused into the vasculature. This erythrocyte adherence was accompanied by a higher increment in vascular resistance during the passage of RBCs through the microcirculation, and by a delay in the recovery toward baseline. The erythrocyte adherence in desmopressin-treated microvasculature was completely abolished with antibodies to von Willebrand factor (vWF). Desmopressin infusion in rats resulted in elevated vWF antigen levels and the appearance of extra-large molecular weight forms of vWF in plasma. These findings suggest that normal erythrocytes adhere to desmopressin-conditioned microvascular endothelium and that endothelial cell-derived vWF is involved in the erythrocyte-endothelium interaction.

Multimeric composition of endothelial cell-derived von Willebrand factor.

The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC-conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.

Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases.

We have previously found that the von Willebrand factor released by cultured human umbilical vein endothelial cells appeared as a single high molecular weight band in glyoxyl agarose electrophoresis. In the present studies we report that this high molecular weight endothelial cell-derived von Willebrand factor, when incubated with granulocyte lysates, was cleaved into a series of multimers indistinguishable from those seen in normal plasma (or type II von Willebrand disease). This von Willebrand factor-cleaving activity was released from granulocytes by calcium ionophore A23187 but was not detected in cytosolic fractions depleted of granular contents. It was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. This von Willebrand factor-cleaving activity thus provides a possible mechanism for the generation of plasma von Willebrand factor multimers from the high molecular weight form of von Willebrand factor secreted by endothelial cells.

Safety of invasive procedures in patients with the coagulopathy of liver disease.

To assess the need for prophylactic fresh frozen plasma in patients with chronic liver disease before procedures, we followed 39 consecutive patients with prothrombin times (PT) of 15-29 s who had 71 invasive procedures. A total of 57 procedures was done in 30 patients receiving no blood product support, while 12 patients received blood products within 12 h of 14 procedures. Only three of the latter group received fresh frozen plasma prophylactically to improve the PT. The two groups were similar in the severity of their liver disease. There were nine surgical procedures as well as paracenteses, thoracenteses, lumbar punctures, and central venous line placement. Three bleeding episodes occurred. Two of the bleeding episodes required no further treatment. Because of the low incidence of bleeding and the ease in controlling the bleeding once it occurred, fresh frozen plasma is not recommended for prophylaxis of an elevated prothrombin time.