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The occurrence of AIDS-associated non-Hodgkin's lymphoma (AIDS-NHL) in hemophiliacs may be increasing in the later stages of AIDS diagnoses and may be linked with longer periods of immunosuppression. Of 33 HIV-infected hemophiliacs, those with NHL as their second AIDS-related disease were older and had lower CD4 counts following longer phases of immunosupression compared to those with NHL as their first AIDS-related disease. Antiretroviral therapy was responsible for an increase in survival rate as well as the prolonged immunosupression.

Kindlin‐3 is essential for supporting the bidirectional signaling of integrin αIIbβ3 in platelets by bridging the crosstalk between integrin αIIbβ3 and the cytoplasmic signaling adaptors.

As part of a prospective cohort study initiated in 1983, the human immunodeficiency virus type 1 (HIV-1) status has been periodically determined for patients with clotting disorders (hemophilia A or B, von Willebrand's disease, miscellaneous). The University of North Carolina Hospitals has conducted comprehensive surveillance for nosocomial infections (NI) using modified Centers for Disease Control criteria since 1980 and entered this information in a computerized data base. Cross-matching of our NI data base and hemophiliac/HIV-1 study data base for the time period 1980-1989 revealed that 13 NI occurred in 11 patients during 659 hospitalizations (5,723 hospital days). NI rates per 100 admissions (per 1,000 hospital days) by HIV-1 status were as follows: HIV-1 negative - 0.91 (1.18), HIV-1 positive pre-AIDS = 1.65 (1.84), and AIDS = 6.67 (6.48). NI occurred with a similar frequency in HIV-1 positive pre-AIDS hemophiliacs and HIV-1 negative hemophiliacs (Fisher's exact test, p >0.10). However, NI occurred more frequently in hemophiliacs with AIDS versus HIV-1 positive or negative hemophiliacs (Fisher's exact test, p, A nosocomial infection (NI) is one that a patient acquires while in the hospital. Studies have shown that hospitalized patients with severe illnesses have a high risk of developing NIs. Also, it is well known that patients with AIDS are at risk for developing life-threatening infections. It is estimated that 35 to 70 percent of the people who have hemophilia (a disease in which the blood does not clot properly) test positive for the human immunodeficiency virus type 1 (HIV-1, the virus that causes AIDS). Therefore, a study was performed to determine the prevalence of NIs in hemophiliacs with and without HIV-1 infections. The study included 195 patients with hemophilia. Of these patients, 24 had AIDS, 104 had HIV-1 infections but had not developed symptoms of AIDS (HIV-positive), and 67 did not have an infection with HIV-1 (HIV-negative). The AIDS patients spent a longer time in the hospital (10.3 days) than those who were HIV-positive (9.0 days) or HIV-negative (7.7 days). During the study period, 11 patients got a total of 13 NIs. Blood and urinary tract infections were the most common types of infections. Of the 11 patients who developed NIs, four had AIDS, five were HIV-positive, and two were HIV-negative. These results translate to a NI rate of one out of every 15 hemophiliacs with AIDS, one out of every 61 who are HIV-positive, and one out of every 110 who are HIV-negative. Factors that increased the risk of NI in patients with AIDS included treatment with antibiotics and the use of central intravenous catheters. It is concluded that full-blown AIDS increases the risk of developing NIs, but HIV-1 infections that have not progressed to AIDS do not significantly increase the risk of NI. (Consumer Summary produced by Reliance Medical Information, Inc.)

Patients who test positive for the human immunodeficiency virus and have gone on to develop either acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) are known to have a high incidence of bacterial infections. It is not known if patients who are HIV-positive but have not developed ARC or AIDS are also at increased risk of infection. It is estimated that there are about 1.5 million such people in the United States. There is concern about the risks involved in operating on these asymptomatic patients, because many have decreased immune function at a subclinical level, i.e. detectable only with laboratory or other testing. A study was undertaken to compare the operative risks of two groups of hemophiliacs, one group HIV-positive and the other HIV-negative. A retrospective review identified 102 hemophiliac patients who had undergone surgery; these patients were cross-referenced to patients in an ongoing study of hemophiliac patients who are HIV-positive. This cross-referencing revealed 83 patients with known HIV status who had undergone 169 operations. These patients were grouped according to HIV-positive status, HIV-negative status, or unclear HIV status (those patients who had surgery before their HIV status had been established). The wound infection rates of the three groups were: HIV-positive, 1.4 percent; unclear HIV status 1.5 percent; HIV-negative 0 percent. There was no significant difference in wound infection rates. It is concluded that surgery in HIV-positive, non-AIDS patients, does not carry an increased risk of wound infection. (Consumer Summary produced by Reliance Medical Information, Inc.)

Patients who later go on to develop acquired immunodeficiency syndrome (AIDS) may harbor the human immunodeficiency virus (HIV) in their blood for several years. Hemophilia has been associated with AIDS since 1984; many hemophiliacs were infected by HIV via blood transfusions before the blood supply was screened for this virus. The effect of the compromise of the immune system caused by HIV infection on the outcome of surgery is not known. This is important in the case of hemophiliacs since patients with severe hemophilia frequently have serious joint disorders and need to undergo orthopedic surgery. In this study, hemophiliac patients who were HIV-positive were evaluated regarding the results of their operations. There were 26 operative procedures performed on these patients; only one patient developed infection, however five patients had abnormal temperature elevation following surgery. Their increased temperatures occurred without the expected elevation in white blood cell count. The patients who experienced fever had significantly reduced numbers of CD4 lymphocytes, a type of white blood cell, before surgery. In most cases the HIV infection progressed, with six patients being diagnosed with AIDS. Those patients who had low preoperative CD4 counts appeared to progress more rapidly to AIDS. It should be noted that these values typically vary for the same patient and they should be used only as a guideline. Skin antigens also give an evaluation of CD4 lymphocyte function. The authors suggest that evaluation by CD4 lymphocyte count and skin antigen testing be done to assess immune function prior to elective surgery in HIV-positive patients. Knowledge of how well the immune system is functioning can help predict the risk of infection after surgery. (Consumer Summary produced by Reliance Medical Information, Inc.)

Kindlin-3, a protein 4.1, ezrin, radixin, and moesin (FERM) domain–containing adaptor in hematopoietic cells, is essentially required for supporting the bidirectional integrin αIIbβ3 signaling in platelets by binding to the integrin β3 cytoplasmic tail. However, the structural details of kindlin-3’s FERM domain remain unknown. In this study, we crystalized the kindlin-3’s FERM domain protein and successfully solved its 3-dimensional structure. The structure shows that the 3 kindlin-3’s FERM subdomains (F1, F2, and F3) compact together and form a cloverleaf-shaped conformation, which is stabilized by the binding interface between the F1 and F3 subdomains. Interestingly, the FERM domain of kindlin-3 exists as a monomer in both crystal and solution, which is different from its counterpart in kindlin-2 that is able to form a F2 subdomain-swapped dimer; nonetheless, dimerization is required for kindlin-3 to support integrin αIIbβ3 activation, indicating that kindlin-3 may use alternative mechanisms for formation of a functional dimer in cells. To evaluate the functional importance of the cloverleaf-like FERM structure in kindlin-3, structure-based mutations were introduced into kindlin-3 to disrupt the F1/F3 interface. The results show that integrin αIIbβ3 activation is significantly suppressed in platelets expressing the kindlin-3 mutant compared with those expressing wild-type kindlin-3. In addition, introduction of equivalent mutations into kindlin-1 and kindlin-2 also significantly compromises their ability to support integrin αIIbβ3 activation in CHO cells. Together, our findings suggest that the cloverleaf-like FERM domain in kindlins is structurally important for supporting integrin αIIbβ3 activation.

•Patients with severe COVID-19 make prothrombotic antibodies linked to platelet activation, inflammation, and tissue damage markers.•A subset of antibodies to the RBD of the spike protein contributes to the pool of prothrombotic antibodies.

Neutrophils fight infections by generating reactive oxygen species (ROS) and extracellular traps (NETs). However, how neutrophils modulate ROS/NET generation is mechanistically unclear. Kindlin‐3, an essential integrin activator expressed in hematopoietic cells, is required to support integrin‐mediated neutrophil recruitment during inflammation. Here, we report a novel role of kindlin‐3 in regulating ROS/NET generation in neutrophils. When overexpressing kindlin‐3 in neutrophil‐like differentiated HL‐60 cells (HL‐60N), ROS/NET generation from these cells were significantly suppressed. Interestingly, overexpression of a kindlin‐3 mutant that is defective for interacting with integrins in HL‐60N cells still inhibited ROS/NET generation, suggesting that the role of kindlin‐3 in inhibiting ROS/NET signaling may be independent of its binding to integrins. Consistently, knockdown of kindlin‐3 in HL‐60N cells led to enhanced ROS/NET generation. In addition, bone marrow neutrophils isolated from kindlin‐3‐deficient mice showed elevated ROS/NET generation when compared with WT counterparts. As expected, overexpression of exogenous kindlin‐3 in mouse neutrophils could suppress NET release ex vivoand in vivo. Collectively, these results demonstrate that kindlin‐3 in neutrophils is involved in modulating the ROS/NET signaling, providing a novel mechanism for fine‐tuning neutrophil behaviors during inflammation. Kindlin‐3 is a novel regulator for fine‐tuning the generation of ROS/NETs in neutrophils during inflammation.

Kindlin-3 essentially supports integrin activation in blood cells. Absence of kindlin-3 in humans causes leukocyte adhesion deficiency-III characterized with severe bleeding disorder and recurrent infections. Previously, we generated kindlin-3 knock-in (K3KI) mice carrying an integrin-interaction disrupting mutation in kindlin-3 and verified the functional significance of the binding of kindlin-3 to integrin αIIbβ3 in platelets. Here, using K3KI mice, we functionally evaluate the crosstalk between kindlin-3 and β2-integrins in neutrophils. Although the kindlin-3 mutant in K3KI neutrophils is normally expressed, its binding ability to β2-integrins in neutrophils is disabled. In vitro and in vivo analyses disclose that β2-integrin–mediated K3KI neutrophil adhesion and recruitment are significantly suppressed. Interestingly, the ability of releasing neutrophil extracellular traps (NETs) from K3KI neutrophils is also compromised. Substantially, a peptide derived from the integrin β2 cytoplasmic tail that can inhibit the interaction between kindlin-3 and β2-inegrins significantly jeopardizes NET release without affecting neutrophil adhesion and recruitment under the experimental conditions. These findings suggest that crosstalk between kindlin-3 and β2-integrins in neutrophils is required for supporting both neutrophil recruitment and NET release, but the involved regulatory mechanisms in these two cellular events might be differential, thus providing a novel therapeutic concept to treat innate immune-related diseases.

With advances in care, increasing numbers of people with hemophilia (PWH) achieve near-normal life expectancies and present with typical age-related cardiovascular conditions. Evidence-based guidelines for medical or surgical management of cardiovascular conditions in individuals with hemophilia are limited. Published recommendations exist for the management of some common cardiovascular conditions (eg, ischemic heart disease, atrial fibrillation), but identifying optimal strategies for anticoagulant or antithrombotic therapy constitutes the primary challenge of managing nonoperative cardiovascular disease (CVD) in PWH. In general, as long as factor concentrates or other hemostatic therapies maintain adequate hemostasis, the recommended medical and surgical management of CVD in PWH parallels that in individuals without hemophilia. The presence of factor inhibitors complicates hemophilia management. Published outcomes of CVD treatment in PWH are similar to those in the general population. Specific knowledge about factor replacement, factor inhibitors, and disease-specific treatment distinguishes the cardiovascular care of PWH from similar care of individuals without this rare bleeding disorder. Furthermore, a multidisciplinary approach incorporating a hematologist with an onsite coagulation laboratory, ideally associated with a hemophilia treatment center, is integral to the management of CVD in PWH.

Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive antibodies are central to the pathogenesis of heparin-induced thrombocytopenia (HIT). It is as yet unclear what triggers the initial induction of pathogenic antibodies. We identified B cells in peripheral blood of healthy adults that produce PF4/heparin-specific antibodies following in vitro stimulation with proinflammatory molecules containing deoxycytosine-deoxyguanosine (CpG). Similarly, B cells from unmanipulated wild-type mice produced PF4/heparin-specific antibodies following in vitro or in vivo CpG stimulation. Thus, both healthy humans and mice possess preexisting inactive/tolerant PF4/heparin-specific B cells. The findings suggest that breakdown of tolerance leads to PF4/heparin-specific B-cell activation and antibody production in patients developing HIT. Consistent with this concept, mice lacking protein kinase Cδ (PKCδ) that are prone to breakdown of B-cell tolerance produced anti-PF4/heparin antibodies spontaneously. Therefore, breakdown of tolerance can lead to PF4/heparin-specific antibody production, and B-cell tolerance may play an important role in HIT pathogenesis.

CalDAG‐GEFI regulates neutrophil chemotaxis independent of integrin function by a mechanism that involves F‐actin distribution and cell polarization. Chemotaxis and integrin activation are essential processes for neutrophil transmigration in response to injury. CalDAG‐GEFI plays a key role in the activation of β1, β2, and β3 integrins in platelets and neutrophils by exchanging a GDP for a GTP on Rap1. Here, we explored the role of CalDAG‐GEFI and Rap1b in integrin‐independent neutrophil chemotaxis. In a transwell assay, CalDAG‐GEFI−/−neutrophils had a 46% reduction in transmigration compared with WT in response to a low concentration of LTB4. Visualization of migrating neutrophils in the presence of 10 mM EDTA revealed that CalDAG‐GEFI−/−neutrophils had abnormal chemotactic behavior compared with WT neutrophils, including reduced speed and directionality. Interestingly, Rap1b−/−neutrophils had a similar phenotype in this assay, suggesting that CalDAG‐GEFI may be acting through Rap1b. We investigated whether the deficit in integrin‐independent chemotaxis in CalDAG‐GEFI−/−neutrophils could be explained by defective cytoskeleton rearrangement. Indeed, we found that CalDAG‐GEFI−/−neutrophils had reduced formation of F‐actin pseudopodia after LTB4stimulation, suggesting that they have a defect in polarization. Overall, our studies show that CalDAG‐GEFI helps regulate neutrophil chemotaxis, independent of its established role in integrin activation, through a mechanism that involves actin cytoskeleton and cellular polarization.

The most significant complication of treatment in patients with hemophilia A is the development of alloantibodies that inhibit factor VIII activity. In the presence of inhibitory antibodies, replacement of the missing clotting factor by infusion of factor VIII becomes less effective. Once replacement therapy is ineffective, acute management of bleeding requires agents that bypass factor VIII activity. Long-term management consists of eradicating the inhibitor through immune tolerance. Despite success in the treatment of acute bleeding and inhibitor eradication, there remains an inability to predict or prevent inhibitor formation. Ideally, prediction and ultimately prevention will come with an improved understanding of how patient-specific and treatment-related factors work together to influence anti–factor VIII antibody production.

B-cell development is orchestrated by complex signaling networks. Rap1 is a member of the Ras superfamily of small GTP-binding proteins and has 2 isoforms, Rap1a and Rap1b. Although Rap1 has been suggested to have an important role in a variety of cellular processes, no direct evidence demonstrates a role for Rap1 in B-cell biology. In this study, we found that Rap1b was the dominant isoform of Rap1 in B cells. We discovered that Rap1b deficiency in mice barely affected early development of B cells but markedly reduced marginal zone (MZ) B cells in the spleen and mature B cells in peripheral and mucosal lymph nodes. Rap1b-deficient B cells displayed normal survival and proliferation in vivo and in vitro. However, Rap1b-deficient B cells had impaired adhesion and reduced chemotaxis in vitro, and lessened homing to lymph nodes in vivo. Furthermore, we found that Rap1b deficiency had no marked effect on LPS-, BCR-, or SDF-1–induced activation of mitogen-activated protein kinases and AKT but clearly impaired SDF-1–mediated activation of Pyk-2, a key regulator of SDF-1–mediated B-cell migration. Thus, we have discovered a critical and distinct role of Rap1b in mature B-cell trafficking and development of MZ B cells.

Angiogenesis is the main mechanism of vascular remodeling during late development and, after birth, in wound healing. Perturbations of angiogenesis occur in cancer, diabetes, ischemia, and inflammation. While much progress has been made in identifying factors that control angiogenesis, the understanding of the precise molecular mechanisms involved is incomplete. Here we identify a small GTPase, Rap1b, as a positive regulator of angiogenesis. Rap1b-deficient mice had a decreased level of Matrigel plug and neonatal retinal neovascularization, and aortas isolated from Rap1b-deficient animals had a reduced microvessel sprouting response to 2 major physiological regulators of angiogenesis: vascular endothelial growth factor (VEGF) and basic fibroblasts growth factor (bFGF), indicating an intrinsic defect in endothelial cells. Proliferation of retinal endothelial cells in situ and in vitro migration of lung endothelial cells isolated from Rap1b-deficient mice were inhibited. At the molecular level, activation of 2 MAP kinases, p38 MAPK and p42/44 ERK, important regulators of endothelial migration and proliferation, was decreased in Rap1b-deficient endothelial cells in response to VEGF stimulation. These studies provide evidence that Rap1b is required for normal angiogenesis and reveal a novel role of Rap1 in regulation of proangiogenic signaling in endothelial cells.

Platelets are important for primary hemostasis. When a blood vessel is damaged, platelets adhere to exposed subendothelial connective tissue and form a hemostatic plug. Formation of the plug is contingent upon a series of processes, with adhesion, activation, and aggregation all being involved. Patients with quantitative platelet disorders have reduced numbers of platelets. Patients with qualitative disorders have platelets that exhibit abnormal functioning. Defects that impair function can affect platelet receptors, secretory responses, or intracellular signaling pathways. Examples of qualitative platelet disorders include Glanzmann’s thrombasthenia (GT) and Bernard-Soulier syndrome (BSS). The treatment of platelet disorders is primarily with platelet concentrates. However, in patients with abnormalities of their platelet surface receptors, platelet transfusion may provoke an immune response. Recombinant factor VIIa (rFVIIa) may provide hemostatically effective therapy in such patients.

Integrins mediate the adhesion of cells to each other and to the extracellular matrix during development, immunity, metastasis, thrombosis, and wound healing. Molecular defects in either the α- or β-subunit can disrupt integrin synthesis, assembly, and/or binding to adhesive ligands. This is exemplified by the bleeding disorder, Glanzmann thrombasthenia (GT), where abnormalities of the platelet-specific integrin, αIIbβ3, prevent platelet aggregation following vascular injury. We previously used a retrovirus vector containing a cDNA cassette encoding human integrin β3 to restore integrin αIIbβ3 on the surface of megakaryocytes derived from peripheral blood stem cells of GT patients. In the present study, bone marrow from β3-deficient (β3–/–) mice was transduced with the ITGβ3-cassette to investigate whether the platelet progeny could establish hemostasis in vivo. A lentivirus transfer vector equipped with the human ITGA2B gene promoter confined transgene expression to the platelet lineage. Human β3 formed a stable complex with murine αIIb, effectively restoring platelet function. Mice expressing significant levels of αIIbβ3 on circulating platelets exhibited improved bleeding times. Intravenous immunoglobulin effectively diminished platelet clearance in animals that developed an antibody response to αIIbβ3. These results indicate the feasibility of targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders.

Recombinant factor VIIa was initially developed for the treatment of hemorrhagic episodes in hemophilic patients with inhibitors to factors VIII and IX. After its introduction, it has also been used “off-label” to enhance hemostasis in nonhemophilic patients who experience bleeding episodes not responsive to conventional therapy. Evidence so far indicates that the use of factor VIIa in hemophilic patients with inhibitors is both safe and effective. Anecdotal reports also suggest that the product is safe and effective in controlling bleeding in nonhemophilic patients. However, its use in these conditions has not been approved by the FDA, and conclusive evidence of its effectiveness from controlled clinical trials is not yet available. Several questions pertaining to the use of factor VIIa require further investigation, including the mechanism of action; the optimal dose; definitive indications; ultimate safety; and laboratory tests for monitoring therapy. (Blood. 2004;104:3858-3864)

Calcium-binding domains in the α-subunit of integrins contain a central loop structure. To examine the importance of the loop structure, a series of αIIb mutants containing changes to the calcium-liganding amino acids have been constructed. Significantly, none of the mutant αIIbβ3 complexes was detected on the surface of transfected cells, but mutant pro-αIIb was detected in cell lysates in complex with β3. To study the importance of the regions flanking the second calcium-binding domain for ligand-binding and ligand-binding specificity, three αIIb/α5 chimaeras containing α5 sequences flanking or flanking and including the second calcium-binding domain were constructed. The chimaera containing both α5-flanking regions was not expressed on the cell surface, but FR1 and FR2, substituting either the first or second flanking region, were expressed. FR1β3-transfected cells lost the ability to adhere to fibrinogen and to support aggregation and had minimal fibrinogen-binding ability. The heterodimer complex was less stable than the wild-type. FR2β3-transfected cells adhered to fibrinogen and bound soluble fibrinogen with higher affinity when compared with wild-type. In addition, the heterodimer complex was more stable than wild-type. These results indicate that the conformation of the second calcium-binding domain is critical for maturation of the αIIbβ3 complex and expression on the cell surface and that the surrounding sequences are critical for αIIbβ3 function.

Chronic joint disease from repeated bleeding into joints is a serious complication of hemophilia. To measure the extent of and to identify risk factors for deviations from normal in joint range of motion (ROM), we used cross-sectional data collected from 4343 males with hemophilia aged 2 to 19 years who received care at 136 US hemophilia treatment centers (HTCs). Factors examined included age, race/ethnicity, family history, insurance status, age at diagnosis and first HTC visit, frequency of HTC visits, hemophilia type, bleeding frequency, prophylaxis use, inhibitor status, body mass index (BMI), and recent orthopedic procedures. Trained personnel using a standard protocol obtained ROM measurements on 10 joints (hips, knees, shoulders, elbows, and ankles). Analyses used multiple linear regression to model overall ROM limitation separately by disease severity. For persons in all severity groups, joint ROM limitation was positively associated with older age, nonwhite race, and increased BMI. For those with severe disease, ROM limitation was also positively associated with number of bleeds and was greater for those with inhibitors or recent orthopedic procedures. We conclude that ROM limitations begin at an early age, especially for those with severe and moderate disease, and that BMI is an important, potentially modifiable risk factor.

In a phase 1 dose escalation study, 13 subjects with hemophilia A received by peripheral intravenous infusion a retroviral vector carrying a B-domain–deleted human factor VIII (hFVIII) gene. Infusions were well tolerated. Tests for replication competent retrovirus have been negative. Polymerase chain reaction (PCR) analyses demonstrate the persistence of vector gene sequences in peripheral blood mononuclear cells in 3 of 3 subjects tested. Factor VIII was measured in serial samples using both a one-stage clotting assay and a chromogenic assay. While no subject had sustained FVIII increases, 9 subjects had FVIII higher than 1% on at least 2 occasions 5 or more days after infusion of exogenous FVIII, with isolated levels that ranged from 2.3% to 19%. Pharmacokinetic parameters of exogenous FVIII infused into subjects 13 weeks after vector infusion showed an increased half-life (T1/2; P < .02) and area under the curve (AUC, P < .04) compared with prestudy values. Bleeding frequency decreased in 5 subjects compared with historical rates. These results demonstrate that this retroviral vector (hFVIII(V)) is safe and, in some subjects, persists more than a year in peripheral blood mononuclear cells, with measurable factor VIII levels and with increased available FVIII activity (increased T1/2 and AUC) after infusion of exogenous FVIII concentrate.

Gene transfer is an exciting and potentially important treatment approach for hemophilia A and B. Four phase I clinical trials of the safety of gene transfer in hemophilia A or B have been completed and two more trials are currently underway. The results of these trials indicate that gene transfer in hemophilia with the vectors and doses used is safe and well tolerated. Efforts continue to understand the basic biology and improve the efficiency of gene transfer.

Many persons with hemophilia were infected with hepatitis C and B viruses (HCV, HBV) and HIV, but the consequences of these transfusion-acquired infections are poorly defined. We estimated the risk of HCV-related end-stage liver disease (ESLD) and the associations of age, HBV, and HIV with that risk. All 1816 HCV-seropositive hemophilic patients at 16 centers were followed for up to 16 years. Of these, 624 were HIV− and 1192 were HIV-coinfected; 135 had persistent HBV surface antigenemia, 1374 had resolved HBV infection, and 287 were HBV-uninfected. ESLD was defined as bleeding esophageal varices, hepatic encephalopathy, persistent ascites, or death excluding nonhepatic causes of these conditions. Competing risk models were used to estimate the annual hazard rate and cumulative incidence of ESLD. Proportional hazards models were used to estimate relative hazards of ESLD with covariates. ESLD developed in 127 of the HCV/HIV-coinfected participants, with an estimated 16-year cumulative incidence of 14.0% (95% confidence interval [CI], 11.6%-16.4%). Without HIV, 10 HCV-infected participants developed ESLD, for a significantly lower cumulative incidence of 2.6% (95% CI, 1.0%-4.3%,P < .0001). ESLD risk increased steeply with age in both groups. With HIV, ESLD risk was increased 8.1-fold (95% CI, 1.9-35.2) with HBV surface antigenemia, 2.1-fold (95% CI, 1.3-3.3) with fewer than 0.2 × 109/L (200/μL) CD4+lymphocytes, and 1.04-fold (95% CI, 1.03-1.06) per year of age. Thus, HIV is associated with a markedly increased risk of HCV-related ESLD for persons with hemophilia, particularly with HBV infection, low CD4+ lymphocytes, or older age.

The affinity of integrin αIIbβ3for fibrinogen is controlled by inside-out signals that are triggered by agonists like thrombin. Agonist treatment of platelets also activates Rap1b, a small GTPase known to promote integrin-dependent adhesion of other cells. Therefore, we investigated the role of Rap1b in αIIbβ3function by viral transduction of GFP-Rap1 chimeras into murine megakaryocytes, which exhibit inside-out signaling similar to platelets. Expression of constitutively active GFP-Rap1b (V12) had no effect on unstimulated megakaryocytes, but it greatly augmented fibrinogen binding to αIIbβ3induced by a PAR4 thrombin receptor agonist (p< 0.01). The Rap1b effect was cell-autonomous and was prevented by pre-treating cells with cytochalasin D or latrunculin A to inhibit actin polymerization. Rap1b-dependent fibrinogen binding to megakaryocytes was blocked by POW-2, a novel monovalent antibody Fab fragment specific for high affinity murine αIIbβ3. In contrast to GFP-Rap1b (V12), expression of GFP-Rap1GAP, which deactivates endogenous Rap1, inhibited agonist-induced fibrinogen binding (p< 0.01), as did dominant-negative GFP-Rap1b (N17) (p< 0.05). None of these treatments affected surface expression of αIIbβ3. These studies establish that Rap1b can augment agonist-induced ligand binding to αIIbβ3through effects on integrin affinity, possibly by modulating αIIbβ3interactions with the actin cytoskeleton.

Mechanisms that protect most high-risk HIV-1 seronegative (HRSN) persons are not well understood. Among hemophiliacs from the Multicenter Hemophilia Cohort Study who remained HIV-1 seronegative despite a high (94%) risk for acquisition of HIV-1 infection, only 7/43 were homozygous for the protective CCR5 Δ32 polymorphism. Among the remainder, neither CCR5 density nor β-chemokine production, nor in vitro susceptibility to infection with the HIV-1 isolate JR-FL could distinguish HRSN hemophiliacs from healthy controls. When compared to lymphocytes of healthy controls not at risk for HIV-1 infection, diminished spontaneous lymphocyte proliferation was seen in lymphocytes of HRSN hemophiliacs as well as in lymphocytes of hemophiliacs not at risk for HIV-1 infection. Surprisingly sera/plasmas obtained from high-risk HIV-1 seropositve hemophiliacs prior to seroconversion more often contained alloreactive antibodies than date-matched sera/plasmas obtained from HRSN hemophiliacs. Thus alloreactivity may predispose to acquisition of HIV-1 infection after parenteral exposure.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, ?IIbß3. As a result, ?IIbß3 cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889PlA2ß3, was transduced into peripheral blood CD34+ cells from 2 patients with thrombasthenia with defects in the ß3 gene. The human ?IIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type ß3 subunit. Proviral DNA and ?IIbß3 biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34+ cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed ?IIbß3 on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the ?IIbß3 complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34+ cells as targets for ?IIb promoter-driven MuLV vectors for gene therapy of platelet disorders.

To further identify amino acid domains involved in the ligand binding specificity of αIIbβ3, chimeras of the conserved calcium binding domains of αIIband the α subunit of the fibronectin receptor α5β1were constructed. Chimeras that replaced all four calcium binding domains, replaced all but the second calcium binding domain of αIIbwith those of α5, or deleted all four calcium binding domains were synthesized but not expressed on the cell surface. Additional chimeras exchanged subsets or all of the variant amino acids in the second calcium binding domain, a region implicated in ligand binding. Cell surface expression of each second calcium binding domain mutant complexed with β3was observed. Each second calcium binding domain mutant was able to 1) bind to immobilized fibrinogen, 2) form fibrinogen-dependent aggregates after treatment with dithiothreitol, and 3) bind the activation-dependent antibody PAC1 after LIBS 6 treatment. Soluble fibrinogen binding studies suggested that there were only small changes in either the KdorBmaxof any mutant. We conclude that chimeras of αIIbcontaining the second calcium binding domain sequences of α5are capable of complexing with β3, that the complexes are expressed on the cell surface, and that mutant complexes are capable of binding both immobilized and soluble fibrinogen, suggesting that the second calcium binding domain does not determine ligand binding specificity.

Combined factors V and VIII deficiency is an autosomal recessive bleeding disorder associated with plasma levels of coagulation factors V and VIII approximately 5% to 30% of normal. The disease gene was recently identified as the endoplasmic reticulum-Golgi intermediate compartment protein ERGIC-53 by positional cloning, with the detection of two founder mutations in 10 Jewish families. To identify mutations in additional families, the structure of the ERGIC-53 gene was determined by genomic polymerase chain reaction (PCR) and sequence analysis of bacterial artificial chromosome clones containing the ERGIC-53 gene. Nineteen additional families were analyzed by direct sequence analysis of the entire coding region and the intron/exon junctions. Seven novel mutations were identified in 10 families, with one additional family found to harbor one of the two previously described mutations. All of the identified mutations would be predicted to result in complete absence of functional ERGIC-53 protein. In 8 of 19 families, no mutation was identified. Genotyping data indicate that at least two of these families are not linked to the ERGIC-53 locus. Taken together, these results suggest that a significant subset of combined factors V and VIII deficiency is due to mutation in one or more additional genes.

• We treated 193 patients either intravenously (94) or subcutaneously (99) for at least 5 days with porcine intestinal mucosal heparin and followed them up prospectively with frequent platelet counts to determine the incidence of heparin-related thrombocytopenia and arterial thrombosis. None of the patients in the study developed severe thrombocytopenia (platelet count, 100 × 109/L) or arterial thrombosis. Eight patients had a platelet count of 100 to 140×109/L on one occasion, with a count of 140 × 109/L on the subsequent measurement. The mean (± SD) values of the initial and lowest platelet counts during therapy in all patients were 288±100×109/L and 253±88× 109/L, respectively, with the lowest counts occurring on day 4.1 ± 4.2. A least-squares line was computed for each patient to fit the day and counts; the slopes were significantly different from zero and negative in 7.8% of patients and positive in 14.5%. This multicenter study confirms the reports that the incidence of heparinrelated severe thrombocytopenia and arterial thrombosis is distinctly low in patients treated with porcine-mucosal heparin.(Arch Intern Med. 1989;149:1285-1288)

• Cofactors for the clinical expression of infection due to the human immunodeficiency virus (HIV) are not well understood. We asked if there was a familial tendency to the development of complications of HIV infection. We examined 35 hemophilic sibships in which at least two brothers with classic hemophilia (factor VIII deficiency) were infected with HIV. Twenty-four (34%) of the 70 patients had serious sequelae of infection, and 46 (66%) were asymptomatic or had only lymph node enlargement. Using Fisher's exact test, we found the concordance among siblings for serious sequelae of HIV infection was greater than would be expected by chance. When analysis was restricted to include only siblings known to be infected for more than two years, this concordance was still present. In the study population, birth order and mean yearly usage of factor VIII concentrate were unrelated to the outcome of HIV infection. The data indicate a familial tendency to serious complications of HIV infection. The factor(s) responsible for this familial tendency are currently under investigation.(Arch Intern Med 1989;149:885-888)

Rap1B is a low molecular weight G protein which is phosphorylated by cAMP-dependent protein kinase. In order to identify the site of phosphorylation by cAMP-dependent protein kinase, purified rap1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed-phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was -Lys-Lys-Ser-Ser-. This sequence is near the carboxyl terminus and is adjacent to the site of membrane attachment of the protein.

To determine the relationship between equilibrium binding of thrombin to sites on the platelet surface and the cleavage of membrane glycoprotein V (GPV) by thrombin, we examined the effect of active site-modified thrombin (1 -chloro-3-tosylamido-7-amino-l-2-heptanone thrombin toslysCH2-thrombin) on the binding of native thrombin to platelets and on the hydrolysis of GPV by native thrombin. ToslysCH2-thrombin inhibited binding of native thrombin to high affinity sites on the platelet surface. In contrast, hydrolysis of GPV by native thrombin, even at threshold thrombin concentrations, was not inhibited by pretreatment with toslysCH2-thrombin at concentrations up to 210 nmol/L. ToslysCH2-thrombin also had no appreciable effect on platelet aggregation or release of 14C-serotonin induced by native thrombin. Because toslysCH2-thrombin does not inhibit platelet release, aggregation, or GPV hydrolysis by native thrombin but does inhibit high affinity surface binding by native thrombin, these results indicate that thrombin binding and hydrolysis of GPV are separate and unrelated events.

The clinical manifestations of severe preeclampsia and thrombotic thrombocytopenic purpura are similar. Patients with thrombotic thrombocytopenic purpura have been demonstrated to have larger than usual von Willebrand factor multimers. Serial serum samples were taken from patients with severe preeclampsia, all with platelet counts less than 50,000. Von Willebrand factor multimeric patterns were normal in all the patients except one, who later proved to have chronic relapsing thrombotic thrombocytopenic purpura. All patients had elevated levels of von Willebrand factor. Although the clinical presentations of thrombotic thrombocytopenic purpura and severe preeclampsia with thrombocytopenia have many similarities, the underlying pathophysiology of each disorder appears to be different.

Daily administration of large doses of factor VIII concentrate in a hemophiliac with a high titer factor VIII inhibitor resulted in marked reduction in the titer and response of the inhibitor to factor VIII administration and made possible elbow and bilateral knee replacements under conventional factor VIII coverage. Studies performed during the course of treatment indicated that the reduction in the inhibitor was the result of specific tolerance to factor VIII.

To further elucidate the natural history of human immunodeficiency virus (HIV) infection, we studied intermediate HIV-related conditions occurring before acquired immunodeficiency syndrome (AIDS) in a prospectively observed multicenter cohort of 738 HIV-infected persons with hemophilia. We analyzed the frequency in adults and children of common HIV-related conditions and the relative risk of AIDS after occurrence of these conditions, controlling for age at seroconversion and the percentage of CD4+lymphocytes. Thrombocytopenia was the most frequently observed condition with cumulative incidences of 43% ± 7% in adults and 27% ± 6% in children and adolescents by 10 years after seroconversion. Oral candidiasis, fever, weight loss, and non-AIDS pneumonia were two to four times more common in adults than children, whereas herpes zoster risk was similar in the two age groups. HIV-related conditions were infrequent during the first 4 years of infection, particularly in children. With the exception of thrombocytopenia, mean CD4 counts were less than 350 cells/μL at the onset of the conditions. The relative hazard of AIDS after oral candidiasis was 18 in children and 3.8 in adults. Relative hazard in adults was also increased after persistent fever (10), weight loss (3.2), and non-AIDS pneumonia (2.2). Herpes zoster and thrombocytopenia were not significantly associated with AIDS in either age group. We conclude that intermediate HIV-related conditions occur more frequently in adults than in children with hemophilia. Persistent fever is the strongest predictor of AIDS in adults, whereas oral candidiasis is the strongest predictor in children. These findings should facilitate the design and conduct of clinical trials as well as the management of HIV-infected children and adults.

Integrin αIIbβ3functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either “inside-out” or “outside-in” signaling through αIIbβ3is the phosphorylation of focal adhesion kinase (pp125FAK) and the association of pp125FAKwith cytoskeletal components. To examine the role of pp125FAKin these integrin-mediated events, pp125FAKphosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of αIIbβ3: αIIbβ3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125FAKis altered, and αIIbβ3(F727A/K729E/F730A), in which the putative binding site for α-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing αIIbβ3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing αIIbβ3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125FAKis recruited to focal adhesions in adherent cells expressing αIIbβ3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing αIIbβ3(F727A/K729E/F730A), pp125FAKwas phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125FAKcan be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.

Metalloproteases from at least two Gram-negative organisms selectively hydrolyze the surface of human fixed washed platelets. The protease-treated platelets lose their ability to aggregate with bovine von Willebrand factor and human von Willebrand factor plus ristocetin. The present study reports the membrane glycoprotein alterations and loss of platelet function that occur after incubation of fresh washed human platelets with a purified protease produced by Serratia marcescens. The studies were undertaken in order to examine, using the Serratiaprotease as an enzymatic probe, the relationship between externally oriented glycoproteins and two known aggregating agents, bovine von Willebrand factor and thrombin. Platelet membrane glycoproteins were analyzed after discontinuous NaDodSO4/polyacrylamide gel electrophoresis using both staining with periodic acid-Schiff reagent and autoradiography of 3H-labeled platelets. Both methods of detection demonstrated that at Serratiaprotease concentrations above 0.6 μg/ml there was hydrolysis within 3 min of a membrane glycoprotein (Mr185,000) corresponding to GPIb in control platelets. The loss of GPIb was accompanied by the appearance in the platelet supernatant of a glycopeptide (Mr156,000). Under reducing conditions, the hydrolyzed membrane glycopeptide in the supernatant had a consistently faster migration (Mr149,000). Platelets treated with Serratiaprotease at concentrations sufficient to give maximal cleavage of GPIb were unresponsive to bovine von Willebrand factor and did not aggregate or release [14C]serotonin at doses of von Willebrand factor up to 0.33 unit/ml. On the other hand, the response to bovine α-thrombin was only minimally impaired by treatment with Serratiaprotease. These results implicate GPIb in the response by platelets to bovine von Willebrand factor but suggest that surface components other than GPIb play a major role in the response by platelets to thrombin.

Replacement therapy for hemophilia B (factor IX deficiency) using prothrombin complex concentrate (PCC) has been associated with serious complications of thromboembolic events and transmission of viral infections. Monoclonal anti-body-purified factor IX (Mononine) provides a highly purified factor IX concentrate, while eliminating other vitamin K-de-pendent factors (II, VII, and X). Mononine was evaluated for in vivo recovery, half-life, and for its safety and efficacy in 10 patients with hemophilia B. The in vivo recovery of factor IX with Mononine was a 0.67 ± 0.14 U/dL (mean ± SD) increase per 1 U/kg of infused factor IX, and the biologic half-life (t1/2), determined using the terminal phase of elimination, was 22.6 ± 8.1 hours. Comparison of in vivo recovery of other vitamin K-dependent factors following a single infusion of either Mononine or PCC showed that, whereas Mononine infusion caused no changes in other vitamin K-dependent factors or in prothrombin activation fragment (F1+2), PCC infusion was associated with significant increases of factors II (2.7 U/dL per 1 U/dL of IX increase) and X (2.2 U/dL for 1 U/dL of IX). Patients who used Mononine as their sole therapeutic material during the 12-month period showed an excellent response in hemostasis for their bleeding episodes. Their experience with long-term use of Mononine was at least equivalent to their previous experience with PCC in the frequency and amount of factor usage. No patients developed antibody against mouse IgG or an increase in IX inhibitor during the 12-month period. These results indicate that monoclonal antibody-purified factor IX concentrate provides hemostatically effective factor IX replacement while avoiding extraneous thrombogenic substances.© 1992 by The American Society of Hematology. 0006-4971/92/7903-0020$3.00/0

Factors affecting the coagulant activity of two different prothrombin complex concentrates have been investigated using a sensitive in vitro assay developed in this laboratory. One concentrate contained substantial amounts of potentially thrombogenic material, while the other, which was deliberately fortified with antithrombin III and heparin during production, was judged to be relatively nonthrombogenic. The coagulant activity of the thrombogenic concentrate has been partially identified and was due largely to the presence of coagulation factors IXa and Xa. Neither concentrate contained detectable thrombin. However, after incubation with calcium or various poly-amines, large amounts of additional coagulant material, including thrombin, appeared. Heparin and antithrombin III not only neutralized the thrombogenic materials present in the thrombogenic concentrate, but also inhibited the de novo generation of coagulant enzymes during incubation with calcium. The implication of these studies on the preparation of prothrombin complex concentrates and on host susceptibility to thrombosis during the clinical use of these concentrates is discussed.

Studies in several laboratories have suggested that platelets from patients with Glanzmann’s thrombasthenia are deficient in two major membrane glycoproteins and that this membrane defect is uniform from patient to patient. We have used an improved electrophoretic technique to study further the surface composition of normal and thrombasthenie platelets. Platelets from three unrelated thrombasthenie patients were labeled by either lactoperoxidase-catalyzed iodination or the neuraminidase-galactose oxidase-[3H]NaBH4technique and the labeled proteins were separated by two dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. With both techniques, the major radiolabeled proteins were clearly separated from each other and were present as a horizontal collection of discrete spots that suggest charge heterogeneity. Most of the labeled proteins had an acidic isoelectric point. Compared to normal platelets, platelets from patients with Glanzmann’s disease contained no electrophoretically identifiable fibrinogen. In two patients with thrombasthenia, there was total absence of surface glycoproteins GPIIb and GPIII. while a third patient with thrombasthenia, who was clinically indistinguishable from the previous two patients, had decreased, but detectable, amounts of GPIIb and GPIII. These studies suggest that there are at least two phenotypic patterns of membrane abnormalities in Glanzmann’s thrombasthenia involving GPIIb and GPIII and may indicate genetic heterogeneity in this disease.

Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins. It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets. To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening. We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2. A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21). RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras. Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras. Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells. We conclude that RGL2 may be an effector for Ras and/or Rap proteins.

ABSTRACTIndividuals who are homozygous for the 32-bp deletion in the gene coding for the chemokine receptor and major human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 (CCR5-/-) lack functional cell surface CCR5 molecules and are relatively resistant to HIV-1 infection. HIV-1 infection in CCR5-/- individuals, although rare, has been increasingly documented. We now report that the viral quasispecies from one such individual throughout disease is homogenous, T cell line tropic, and phenotypically syncytium inducing (SI); exclusively uses CXCR4; and replicates well in CCR5-/- primary T cells. The recently discovered coreceptors BOB and Bonzo are not used. Although early and persistent SI variants have been described in longitudinal studies, this is the first demonstration of exclusive and persistent CXCR4 usage. With the caveat that the earliest viruses available from this subject were from approximately 4 years following primary infection, these data suggest that HIV-1 infection can be mediated and persistently maintained by viruses which exclusively utilize CXCR4. The lack of evolution toward the available minor coreceptors in this subject underscores the dominant biological roles of the major coreceptors CCR5 and CXCR4. This and two similar subjects (R. Biti, R. Ffrench, J. Young, B. Bennetts, G. Stewart, and T. Liang, Nat. Med. 3:252–253, 1997; I. Theodoreu, L. Meyer, M. Magierowska, C. Katlama, and C. Rouzioux, Lancet 349:1219–1220, 1997) showed relatively rapid CD4+T-cell declines despite average or low initial viral RNA load. Since viruses which use CXCR4 exclusively cannot infect macrophages, these data have implications for the relative infection of the T-cell compartment versus the macrophage compartment in vivo and for the development of CCR5-based therapeutics.

Plasma levels of antithrombin-heparin cofactor, determined by heparin-dependent antithrombin assay, and antithrombin III antigen were measured in 22 members of a large kindred predisposed to venous thrombosis. While 11 members had reduced plasma levels of both antithrombin-heparin cofactor and antithrombin III antigen, the levels of antithrombin-heparin cofactor were always greater than the levels of antithrombin III antigen: 66% (±7%) and 49% (±5%) of normal plasma, respectively. Pooled normal plasma and plasma from one of the affected family members (60% antithrombin-heparin cofactor and 47% antithrombin III antigen) were fractionated by heparin-agarose affinity chromatography. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 0.4 MNaCI and did not cross-react with antibody specific for antithrombin III and did not inhibit factor Xa at an appreciable rate in the presence of heparin, was designated heparin cofactor A. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 2.0 MNaCI. was functionally and antigenically identified as antithrombin III. The concentrations of heparin cofactor A in normal and patient plasma were similar (4.5 × 10−7M).while the concentration of antithrombin III in patient plasma (8.0 × 10−7M) was only 50% of normal (1.6 × 10−6M). The functional properties of both heparin cofactor A and antithrombin III obtained from patient plasma were normal. From the results of the present study it would appear that the antithrombin-heparin cofactor concentration measured in patient plasma reflects the combined concentrations of heparin cofactor A and antithrombin III. Since heparin cofactor A does not cross-react with antibody to antithrombin III, the concentration of antithrombin III antigen in patient plasma is thus lower than the concentration of antithrombin-heparin cofactor.