Two patients with history of STEC-HUS, posttransplant recurrence and complement gene mutations.

Hemolytic uremic syndrome (HUS) is a disease of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. About 90% of cases are secondary to infections by Escherichia coli strains producing Shiga-like toxins (STEC-HUS), while 10% are associated with mutations in genes encoding proteins of complement system (aHUS). We describe two patients with a clinical history of STEC-HUS, who developed end-stage renal disease (ESRD) soon after disease onset. They received a kidney transplant but lost the graft for HUS recurrence, a complication more commonly observed in aHUS. Before planning a second renal transplantation, the two patients underwent genetic screening for aHUS-associated mutations that revealed the presence of a heterozygous CFI mutation in patient #1 and a heterozygous MCP mutation in patient #2, and also in her mother who donated the kidney. This finding argues that the two cases originally diagnosed as STEC-HUS had indeed aHUS triggered by STEC infection on a genetic background of impaired complement regulation. Complement gene sequencing should be performed before kidney transplantation in patients who developed ESRD following STEC-HUS since they may be undiagnosed cases of aHUS, at risk of posttransplant recurrence. Furthermore, genetic analysis of donors is mandatory before living-related transplantation to exclude carriers of HUS-predisposing mutations.

Adenoviral-mediated gene transfer restores plasma ADAMTS13 antigen and activity in ADAMTS13 knockout mice.

ADAMTS13 is a plasma metalloprotease that regulates the size of the von Willebrand factor (VWF) multimers. Genetic or acquired deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP) in humans. Plasma infusion is the treatment of choice for patients with congenital ADAMTS13 deficiency. However, this practice exposes patients to the risk of infections, allergies and fluid volume overload. The search for alternative treatments is required. Here, we tested the ability of systemically administered adenovirus encoding human ADAMTS13 to restore the deficient protein in the circulation of Adamts13(-/-) mice. Injection of the adenovirus efficiently transduced the liver, kidney, lung, heart and spleen, resulting in the secretion of ADAMTS13 into plasma. A reduced area of thrombi was observed when blood from Ad-ADAMTS13-treated mice was perfused over a collagen-coated surface in a parallel plate flow chamber compared with blood of Ad-betaGal-treated controls. The secreted ADAMTS13 protein was functionally active even after 2 months from injection. The data provide the proof of principle for developing a novel therapy for the correction of ADAMTS13 deficiency in patients with hereditary TTP.

Measurement of von Willebrand factor cleaving protease (ADAMTS-13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas.

BACKGROUND: ADAMTS-13 is a von Willebrand factor (VFW)-cleaving protease. Its congenital or acquired deficiency is associated with thrombotic thrombocytopenic purpura (TTP) and more rarely with the hemolytic uremic syndrome. We report on a survey evaluating 11 methods for ADAMTS-13 measurement performed in different labs. DESIGN: Two plasmas, one normal and one from a patient with familial TTP, were mixed at the co-ordinating center to prepare 6 plasmas with 0%, 10%, 20%, 40%, 80% and 100% ADAMTS-13 levels. Each plasma was aliquoted and assembled into sets of 60 (coded from 1 to 60), each containing 10 copies of the original 6 plasmas. Plasmas were frozen and shipped in dry ice to 10 labs with a common frozen reference plasma. Laboratories were asked to measure ADAMTS-13 with their methods. Results were sent to the coordinating center for statistical analysis. RESULTS: Of the 10 methods performed under static conditions 9 were quantitative and one was semiquantitative. One method performed under flow conditions evaluated the extent of cleavage of endothelial cell-derived ultralarge VWF string-like structures and expressed results as deficient, normal, or borderline. Linearity (expected-vs-observed levels), assessed as the squared correlation coefficient, ranged from 0.98 to 0.39. Reproducibility, expressed as the coefficient of variation for repeated measurements, ranged from < 10% to 83%. The majority of methods were able to discriminate between different ADAMTS-13 levels. The majority were able to detect the plasma with 0% level and some of them to discriminate between 0% and 10%. Overall the best performance was observed for three methods measuring cleaved VWF by ristocetin cofactor, collagen binding, and immunoblotting of degraded multimers of VWF substrate, respectively. The poor interlaboratory agreement of results was hardly affected by the use of the common standard. The method performed under flow conditions identified the plasmas with 0%, 10%, 20% and 40% activity as deficient in 7, 5, 1 and 3 of the 10 replicate measurements. The plasmas with 80% and 100% were identified as normal in all of the 10 replicate measurements. CONCLUSIONS: The survey shows varied performance, but supports an optimistic view about the reliability of current methods for ADAMTS-13.

Mecanismos del daño endotelial en la microangiopatía trombótica / Mechanisms of endothelial damage in thrombotic microangiopathy

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Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress.

Verotoxin-1 (VT-1)-producing Escherichia coli is the causative agent of postdiarrheal hemolytic uremic syndrome (D+HUS) of children, which leads to renal and other organ microvascular thrombosis. Why thrombi form only on arterioles and capillaries is not known. This study investigated whether VT-1 directly affected endothelial antithrombogenic properties promoting platelet deposition and thrombus formation on human microvascular endothelial cell line (HMEC-1) under high shear stress. Human umbilical vein endothelial cells (HUVECs) were used for comparison as a large-vessel endothelium. HMEC-1 and HUVECs were pre-exposed for 24 hours to increasing concentrations of VT-1 (2-50 pM) and then perfused at 60 dynes/cm(2) with heparinized human blood prelabeled with mepacrine. Results showed that VT-1 significantly increased platelet adhesion and thrombus formation on HMEC-1 in comparison with unstimulated control cells. An increase in thrombus formation was also observed on HUVECs exposed to VT-1, but to a remarkably lower extent. The greater sensitivity of HMEC-1 to the toxin in comparison with HUVECs was at least in part due to a higher expression of VT-1 receptor (20-fold more) as documented by FACS analysis. The HMEC-1 line had a comparable susceptibility to the thrombogenic effect of VT-1 as primary human microvascular cells of the same dermal origin (HDMECs). The adhesive molecules involved in VT-induced thrombus formation were also studied. Blocking the binding of von Willebrand factor to platelet glycoprotein Ib by aurintricarboxylic acid (ATA) or inhibition of platelet alpha(IIb)beta(3)-integrin by chimeric 7E3 Fab resulted in a significant reduction of VT-1-induced thrombus formation, suggesting the involvement of von Willebrand factor-platelet interaction at high shear stress in this phenomenon. Functional blockade of endothelial beta(3)-integrin subunit, vitronectin receptor, P-selectin, and PECAM-1 with specific antibodies was associated with a significant decrease of the endothelial area covered by thrombi. Confocal microscopy studies revealed that VT-1 increased the expression of vitronectin receptor and P-selectin and redistributed PECAM-1 away from the cell-cell border of HMEC-1, as well as of HDMECs, thus indicating that the above endothelial adhesion molecules are directly involved and possibly determine the effect of VT-1 on enhancing platelet adhesion and thrombus formation in microvascular endothelium. These results might help to explain why thrombi in HUS localize in microvessels rather than in larger ones and provide insights on the molecular events involved in the process of microvascular thrombosis associated with D+HUS.

A novel interpretation of the role of von Willebrand factor in thrombotic microangiopathies based on platelet adhesion studies at high shear rate flow.

Clinical manifestations of thrombotic microangiopathies (TMA) are secondary to platelet aggregation and thrombotic occlusion of the microvasculature of the affected organs. Abnormalities in von Willebrand factor (vWF) in these patients were considered instrumental in promoting the process leading to microvascular thrombosis. We evaluated the capacity of plasma in these patients to induce adhesion of normal platelets and thrombus formation under conditions of controlled fluid shear stress. We also studied vWF multimeric distribution to establish whether abnormalities of this glycoprotein correlate with platelet adhesion and thrombus formation. Plasma from patients in the acute phase and remission showed the same capacity to induce platelet adhesion and thrombus formation at a low level of shear rate (600 sec(-1)) as plasma from control subjects. At a high shear rate (1,500 sec(-1)), platelet adhesion and thrombus dimensions were significantly increased (P: < 0.05) by plasma from patients with TMA compared with controls. The capacity to enhance thrombus formation at high shear stress was present during the acute phase and disease remission and did not correlate with the presence of unusually large vWF multimers. Increased thrombus formation with patient plasma is completely normalized by blocking the interaction of vWF with the platelet receptors, glycoprotein (GP)Ib and GPIIb-IIIa, suggesting that the phenomenon is completely mediated by vWF. Our results suggest the possibility of an intrinsically altered vWF molecule in these patients that is probably more effective than normal vWF in mediating platelet adhesion and thrombus formation.

Increased fragmentation of von Willebrand factor, due to abnormal cleavage of the subunit, parallels disease activity in recurrent hemolytic uremic syndrome and thrombotic thrombocytopenic purpura and discloses predisposition in families. The Italian Registry of Familial and Recurrent HUS/TTP.

We investigated here the changes in von Willebrand factor (vWF) multimers in recurrent, sporadic and familial forms of hemolytic uremic syndrome (HUS)/thrombotic thrombocytopenic purpura (TTP) to see whether they are actually proteolyzed in vivo in these patients. Molecular determinants of fragments in vWF were also characterized to identify possible sites of cleavage of the subunit. Unusually large vWF multimers were found in blood of 8 of 10 patients with recurrent HUS/TTP, both in the acute phase and in remission, but never in familial and sporadic cases. Instead, all of the groups showed evidence of enhanced fragmentation of vWF multimers during the acute phase. Increased fragmentation was also shown by decrease in native 225-kD vWF subunit. In recurrent and sporadic HUS/TTP, enhanced fragmentation normalized at remission, but the abnormality persisted in familial HUS/TTP patients. The latter findings suggest that patients with familial HUS/TTP may have a congenital abnormality in vWF processing. Analysis with specific monoclonal antibodies showed the presence of the normal vWF fragments with apparent molecular mass of 189, 176, and 140 kD in all patients; however, in 6 recurrent and in 5 familial cases, novel fragments that differed in size from normal ones were found. The size of these abnormal fragments differed from one patient to another and none of them was ever found in normal plasma. These results documented, for the first time in HUS/TTP, an abnormal cleavage of the vWF subunit that might account for the increased fragmentation observed in these patients.

Unrecognized pattern of von Willebrand factor abnormalities in hemolytic uremic syndrome and thrombotic thrombocytopenic purpura.

Heterogeneous abnormalities in multimeric structure and fragmentation of endothelial-derived von Willebrand factor (vWF) have been reported in hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). This study was conducted to establish whether different patterns of vWF abnormalities were associated with different clinical syndromes. Plasmatic levels of vWF antigen (vWF:Ag), vWF release from endothelial cells (EC) exposed to patient sera, and vWF multimeric pattern were studied during episodes and again in remission in three groups of patients with severe forms of HUS and TTP paradigmatic of the most common clinical patterns of disease presentation: (1) plasma-responsive; (2) plasma-resistant; and (3) frequently relapsing. Plasma vWF:Ag and serum-induced vWF release from EC were increased in the acute phase of either plasma-responsive and plasma-resistant HUS and TTP, but normalized at remission only in plasma-responsive cases. Both indices were persistently normal in the relapsing forms. Enhanced vWF fragmentation as defined by disappearance of high molecular weight and increase in low molecular weight forms was a consistent finding of the acute phases, and always normalized in remission in all three groups. Unusually large vWF multimers were found exclusively in plasma of relapsing forms of HUS and TTP both during and between relapses. Enhanced levels of vWF:Ag and serum capability to induce vWF release in vitro are markers of disease activity and may reflect systemic endothelial injury and consequent activation. Their presence discriminates acute single-episode cases from relapsing forms and, when failing to normalize with plasma therapy, predicts plasma resistance. Enhanced low molecular weight multimers that closely paralleled disease activity suggest a permissive role of fragmented vWF in the formation of microvascular thrombi. Finally, finding of unusually large multimers exclusively in relapsing forms of HUS and TTP even between relapses, when no other clinical signs of disease activity could be detected, suggests that they cannot be the only factor in microvascular thrombosis.

Neutrophil activation and hemostatic changes in healthy donors receiving granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) enhances neutrophil functions in vitro and in vivo. It is known that neutrophil-derived products can alter the hemostatic balance. To understand whether polymorphonuclear leukocyte (PMN) activation, measured as PMN degranulation and phenotypical change, may be associated to hemostatic alterations in vivo, we have studied the effect of recombinant human G-CSF (rHuG-CSF) administration on leukocyte parameters and hemostatic variables in healthy donors of hematopoietic progenitor cells (HPCs). Twenty-six consecutive healthy donors receiving 10 micrograms/kg/d rHuG-CSF subcutaneously for 5 to 7 days to mobilize HPCs for allogeneic transplants were included in the study. All of them responded to rHuG-CSF with a significant white blood cell count increase. Blood samples were drawn before therapy on days 2 and 5 and 1 week after stopping rHuG-CSF treatment. The following parameters were evaluated: (1) PMN activation parameters, ie, surface CD11b/CD18 antigen expression, plasma elastase antigen levels and cellular elastase activity; (2) plasma markers of endothelium activation, ie, thrombomodulin (TM) and von Willebrand factor (vWF) antigens; (3) plasma markers of blood coagulation activation, ie, F1+2, TAT complex, D-dimer; and (4) mononuclear cell (MNC) procoagulant activity (PCA) expression. The results show that, after starting rHuG-CSF, an in vivo PMN activation occurred, as demonstrated by the significant increment of surface CD11b/CD18 and plasma elastase antigen levels. Moreover, PMN cellular elastase activity, which was significantly increased at 1 day of treatment, returned to baseline at day 5 to 6, in correspondence with the elastase antigen peak in the circulation. This change was accompanied by a parallel significant increase in plasma levels of the two endothelial and the three coagulation markers. The PCA generated in vitro by unstimulated MNC isolated from rHuG-CSF-treated subjects was not different from that of control cells from untreated subjects. However, endotoxin-stimulated MNC isolated from on-treatment individuals produced significantly more PCA compared with both baseline and control samples. All of the parameters were decreased or normal 1 week after stopping treatment. These data show that rHuG-CSF induces PMN activation and transiently affects some hemostatic variables in healthy HPC donor subjects. The clinical significance of these findings remains to be established.

von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.

BACKGROUND: Thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome are severe microvascular disorders of platelet clumping with similar signs and symptoms. Unusually large multimers of von Willebrand factor, capable of agglutinating circulating platelets under high shear stress, occur in the two conditions. We investigated the prevalence of von Willebrand factor-cleaving protease deficiency in patients with familial and nonfamilial forms of these disorders. METHODS: Plasma samples were obtained from 53 patients with thrombotic thrombocytopenic purpura or hemolytic-uremic syndrome. Von Willebrand factor-cleaving protease was assayed in diluted plasma samples with purified normal von Willebrand factor as the substrate. The extent of the degradation of von Willebrand factor was assessed by electrophoresis in sodium dodecyl sulfate-agarose gels and immunoblotting. To determine whether an inhibitor of von Willebrand factor-cleaving protease was present, we measured the protease activity in normal plasma after incubation with plasma from the patients. RESULTS: We examined 30 patients with thrombotic thrombocytopenic purpura and 23 patients with the hemolytic-uremic syndrome. Of 24 patients with nonfamilial thrombotic thrombocytopenic purpura, 20 had severe and 4 had moderate protease deficiency during an acute event. An inhibitor found in 20 of these patients was shown to be IgG in five of five tested plasma samples. Of 13 patients with nonfamilial hemolytic-uremic syndrome, 11 had normal levels of activity of von Willebrand factor-cleaving protease during the acute episode, whereas in 2 patients, the activity was slightly decreased. All 6 patients with familial thrombotic thrombocytopenic purpura lacked von Willebrand factor-cleaving protease activity but had no inhibitor, whereas all 10 patients with familial hemolytic-uremic syndrome had normal protease activity. In vitro proteolytic degradation of von Willebrand factor by the protease was studied in 5 patients with familial and 7 patients with nonfamilial hemolytic-uremic syndrome and was normal in all 12 patients. CONCLUSIONS: Nonfamilial thrombotic thrombocytopenic purpura is due to an inhibitor of von Willebrand factor-cleaving protease, whereas the familial form seems to be caused by a constitutional deficiency of the protease. Patients with the hemolyticuremic syndrome do not have a deficiency of von Willebrand factor-cleaving protease or a defect in von Willebrand factor that leads to its resistance to protease.

Fluid shear stress modulates von Willebrand factor release from human vascular endothelium.

Fluid shear stress generated by blood flow on arterial wall may play a role in the process of atherosclerosis, not only affecting the mass transport phenomena that take place in blood, but also by modulation of synthesis and secretion of humoral factors released by vascular endothelium that mediate platelet-vessel wall interactions. The present study was designed to investigate whether shear stress, induced by laminar flow, modulates von Willebrand factor (vWF) release from cultured human umbilical vein endothelial cells (HUVEC) and whether this physical stimulation can affect vWF synthesis. Monolayers of HUVEC were exposed to laminar flow of varying magnitude (from 2 to 12 dynes/cm2) using a cone-and-plate device. The release of vWF in cell supernatant and in extracellular matrix by cells exposed to flow or maintained in static conditions was evaluated by enzyme-linked immunosorbent assay. HUVEC exposed to laminar flow released higher amounts of vWF into the cell supernatant within few hours of exposure and vWF secretion was dependent on shear stress magnitude. vWF released in extracellular matrix was also higher in cell monolayers exposed to shear than in static controls. vWF mRNA expression in HUVEC was not affected by exposure of cells to laminar flow, indicating that shear-induced vWF release reflected enhanced secretion without de novo protein synthesis. Immunofluorescence studies showed that the release of vWF is due to exocytosis from Weibel-Palade bodies, the storage organelles of vWF. These data indicate a novel mechanism by which local hemodynamic shear forces modulate endothelial cell function and may play a role in development of arterial thrombotic events.

Bilateral nephrectomy stopped disease progression in plasma-resistant hemolytic uremic syndrome with neurological signs and coma.

Four women were admitted over three years because of anemia and renal failure. They had evidence of hemolytic uremic syndrome (HUS) with severe vascular involvement and glomerular collapse. Despite intensive plasma exchange, all patients developed neurologic signs (with seizures and coma in 2) and papilledema. Three developed refractory hypertension and three required dialysis. All patients had abnormal von Willebrand factor (vWF) fragmentation as reflected by decreased high molecular weight and increased low molecular weight vWF multimers in the circulation. Assuming that the disease was sustained by shear stress-induced abnormal vWF fragmentation in damaged renal microvasculature, bilateral nephrectomy was done. Surgery was followed within two weeks by complete hematologic and clinical remission consistently associated with the restoring of vWF fragmentation pathway to normal. We speculate that in HUS resistant to plasma exchange or infusion, removing the kidneys eliminates a major site of vWF fragmentation, which would limit platelet activation and protect patients from the further spreading of microvascular lesions.

alpha 1-Antitrypsin therapy in a case of thrombotic thrombocytopenic purpura.

In thrombotic thrombocytopenic purpura (TTP), activated leucocytes release elastase which mobilises unusually large von Willebrand factor (vWF) multimers from the endothelium. We investigated the effect of an inhibitor of leucocyte elastase (alpha 1-antitrypsin) on circulating vWF and platelet count in a patient with chronic relapsing TTP. alpha 1-antitrypsin consistently normalised vWF multimeric composition but failed to increase platelet count and induce remission. Plasma cryosupernatant, which never normalised vWF, always induced laboratory and clinical remission within 96 hours. In chronic relapsing TTP, unusually large vWF multimers are not the main cause of intravascular platelet aggregation.

Reversible activation defect of the platelet glycoprotein IIb-IIIa complex in patients with uremia.

Patients with chronic renal failure may experience a bleeding tendency and blood loss after surgical procedures or trauma. Altered platelet function has been indicated as the major cause of uremic bleeding, but its pathogenesis remains to be clarified. In two groups of patients with chronic renal disease of various etiology, the receptor function of glycoprotein (GP) Ib and GP IIb-IIIa complex was studied. Glycoprotein Ib was assessed with both 125I-von Willebrand factor (vWF) and 125I-asialo-vWF binding to platelets. Activation-dependent receptor function of the GP IIb-IIIa complex was studied with 125I-fibrinogen and 125I-vWF binding to washed platelets stimulated with adenosine diphosphate plus epinephrine (10 mumol/L each). Flow cytometric analyses on resting and stimulated platelets were performed using an activation-dependent, anti-GP IIb-IIIa monoclonal antibody (PAC1) as well as an activation-independent antibody (LJ-P1). Binding of PAC1 also was assessed in washed and stimulated platelets and in platelet-rich plasma before and after dialysis. We found that the activation-dependent receptor function of the GP IIb-IIIa complex is defective in uremia, as shown by decreased binding of both vWF and fibrinogen to stimulated platelets. Moreover, binding of the activation-dependent anti-GP IIb-IIIa monoclonal antibody, PAC1, was significantly decreased in uremia compared with that of the activation-independent antibody, LJ-P1. Thus, the number of GP IIb-IIIa receptors expressed on the platelet membrane is normal, but their activation is impaired. In contrast to the functional abnormality of GP IIb-IIIa, the vWF-binding activity of GP Ib was normal.(ABSTRACT TRUNCATED AT 250 WORDS)

[Infusion pre-hydration and a humid room in the prevention of postoperative adhesions]. / Preidratazione infusionale e camera umida nella profilassi delle aderenze postchirurgiche.

The Authors after an overview on the most important causes of postsurgical adhesions, drove their attention to the tissue injury determined, during abdominal surgery, by exposure of pelvic serosa to the humidity of operating area. The AA tested body temperature in the Douglas pouch and under the liver on 30 patients who were operated by conservative surgery (myomectomy) and on 8 patient who underwent laparoscopy. A decrease of one degree in the Douglas pouch after one hour on the patient who had a Pfannestiel incision has been detected. While on the patients with longitudinal laparotomy no difference was evidenced. Serosal biopsies done at the beginning of surgery after pelvis exposure to the external conditions (temperature, humidity) showed a marked phlogosis in the tissue of the patients not treated at all. These tissue injuries were almost absent in the group treated with saline tissue irrigation and absent in the group that had parenteral prehydration, too. The AA suggested the use of the two techniques in conservative surgery in order to obtain in the open abdomen surgery results similar to those of the endoscopic one.

Thrombotic thrombocytopenic purpura: evidence that infusion rather than removal of plasma induces remission of the disease.

Plasma exchange has recently been reported to be more effective than plasma infusion for the treatment of thrombotic thrombocytopenic purpura (TTP). However, in the only available controlled study, the plasma infused during the exchange procedure was three times that given by infusion alone. Here we report the case of a patient with chronic relapsing TTP who had 21 relapsing episodes in the last 3 years. During 18 relapses, infusion of plasma, as infusion alone or in the context of an exchange procedure, invariably induced remission of the disease. By contrast, plasma removal alone (replaced with albumin and saline) was ineffective in three further consecutive relapses so that infusion was eventually necessary to induce remission. We concluded that the effective component of plasma exchange in TTP is infusion, rather than removal of plasma. Unusually large von Willebrand factor (ULvWF) multimers were found during both acute and remission phases, possibly reflecting intravascular leakage from ongoing endothelial cell injury. A relative increase of the 176-kd fragment and a relative decrease of the 225-kd subunit were demonstrated during the acute phase, indicating in vivo proteolytic vWF fragmentation. Since in vitro evidence is available that such fragments of vWF induce platelet aggregation, it is speculated that protease inhibitors of normal plasma help restore normal vWF processing activity in the circulation, which explains remission of the disease associated with the plasma infusion.

Defective platelet aggregation in response to platelet-activating factor in uremia associated with low platelet thromboxane A2 generation.

The bleeding tendency associated with uremia is likely due to a qualitative platelet dysfunction. So far the data available on platelet aggregation are conflicting. Since platelet-activating factor (PAF) plays a role in primary hemostasis, we studied platelet aggregation in response to PAF in 40 patients with chronic uremia on regular hemodialysis and 12 control subjects. Our results showed that in 28 of 40 uremics, platelet aggregation response to PAF was normal, whereas in the remaining 12 it was defective in that no second wave of aggregation was elicited even if the PAF concentrations were increased by a factor of 10,000. This abnormal response was peculiar to PAF and only partially related to factor(s) of plasma origin. The number of platelet PAF receptors and their affinity for the agonist were comparable in controls and "PAF-unresponsive" patients. The defective platelet aggregation in response to PAF was associated with a statistically significant reduction (P less than 0.01) in thromboxane A2 (TxA2) generation in platelet-rich plasma (PRP) challenged with PAF (10 and 100 nmol/L). When PRPs from PAF-unresponsive patients were preincubated with a stable analogue of prostaglandin endoperoxides/TxA2 U-46619, an irreversible platelet aggregation in response to PAF was obtained. Thus in a subpopulation of uremics, platelet aggregation in response to PAF is selectively abnormal as a consequence of a reduced TxA2 generation.

Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit.

In this report we demonstrate that proteolytic cleavage of the constituent subunit is one of the causes determining the heterogeneous size distribution of plasma von Willebrand factor (vWf) multimers. As shown by two-dimensional nonreduced/reduced agarose/polyacrylamide gel electrophoresis, the structure of circulating vWf molecules may deviate from that represented by assemblage of a variable number of identical subunits. Indeed, even though the largest multimers in normal plasma appear to be composed predominantly of intact 225-kD subunits, those of intermediate and smaller size contain also 189-, 176-, and 140-kD proteolytic fragments. Different subunit composition patterns are repeated regularly in multimers of increasing molecular mass, yielding series of bands with similar structure. One of these series consists of molecules without evidence of proteolytic fragmentation, and its smallest member appears to be a dimer of 225-kD subunits. Type IIA von Willebrand disease, characterized by absence of the largest multimers, displays a pattern wherein the fragments of 176 and 140 kD are relatively increased, that of 189 kD is markedly decreased or absent, but the composition of individual multimers is otherwise similar to that of species seen also in normal plasma. In contrast to those in the circulation, all normal platelet vWf multimers contain only intact subunit. These results suggest that proteolytic cleavage of plasma vWf subunits occurs after release from cellular sites, whereas platelet vWf stored in alpha-granules is protected from proteolysis. These findings provide information that may be relevant for understanding the normal processing of vWf multimers and for elucidating the pathogenesis of some of the congenital and acquired structural abnormalities of this molecule.

Functional implications of decreased renal cortical atrial natriuretic peptide binding in experimental diabetes.

Glomerular hyperfiltration in streptozotocin-induced diabetes mellitus in rats may be mediated by atrial natriuretic peptide (ANP). We wanted to evaluate plasma levels of ANP and plasma volume in relation to renal ANP receptor density and affinity in rats 6 weeks after induction of diabetes. Plasma levels of immunoreactive ANP were significantly higher in hyperglycemic diabetic (75.2 +/- 8.3 pg/ml) than in control animals (34.7 +/- 8.1 pg/ml; p less than 0.01). Administration of insulin to keep diabetic rats normoglycemic normalized plasma levels of immunoreactive ANP (30.5 +/- 5.2 pg/ml). In contrast, plasma volume did not show significant differences among the groups (hyperglycemic diabetes, 46.6 +/- 3.8; normoglycemic diabetes, 42.4 +/- 3.2; controls, 43.2 +/- 2.0 ml/kg body wt). No correlation was found between plasma levels of immunoreactive ANP and plasma volume. By autoradiography a significant reduction in the number of renal cortical ANP receptors was observed in hyperglycemic diabetic rats as compared with controls. At variance, ANP receptor affinity did not change either in the cortex or in the medulla in hyperglycemic diabetics in comparison with control animals. The pathophysiological implication of cortical ANP receptor down-regulation was underscored by the blunted response of glomerular filtration rate to ANP infusion in diabetic animals as compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)