Approach to the treatment, characterization and diagnosis of an acquired auto-antibody directed against factors prothrombin, factor X and factor IX: a case report and review of the literature.

Bleeding disorders secondary to acquired non-inhibitory antibodies directed against vitamin K-dependent coagulation proteins are rare. In this report, the authors describe a patient with a low grade lymphoma who presented with a fatal acquired bleeding manifestation and abnormal hemostatic studies resulting from deficiencies in both prothrombin and factor X. Patient plasma samples were collected and studied for the presence of an acquired inhibitor. Levels of plasma coagulation proteins were measured using immunoassay. Patient anti-prothrombin immunoglobulin G was isolated and binding to prothrombin, prothrombin F1.2, factors IX and X was evaluated using immunoblots and competition immunoassay. Prolongation in the prothrombin time and activated partial thromboplastin time suggested a factor deficiency in the common pathway of coagulation. Functional and antigenic levels of both prothrombin and factor X were decreased. An IgG subtype-4 antibody was isolated from patient plasma using affinity chromatography on prothrombin-sepharose. This antibody was found to bind to a common metal-ion-dependent conformational epitope found on the γ-carboxyglutamic acid (Gla) domain of prothrombin, factor X and factor IX. This report represents the first description of an acquired bleeding disorder resulting from a unique cross-reactive auto-antibody against a common metal-ion-dependent antigenic structure on the Gla-domain of the vitamin K-dependent proteins.

The neurovascular unit in the setting of stroke.

Microvessels and neurons respond rapidly and simultaneously in focal regions of ischaemic injury in such a way as to suggest that the responses could be coordinated. The ability of neurons to modulate cerebral blood flow in regions of activation results from neurovascular coupling. But little is known about the microvessel-to-neuron direction of the relationship. The presence and participation of intervening glial cells implies the association of microvessels, glia, and neurons in a 'neurovascular unit'. The interdependent functions of the cellular and matrix components of this theoretical unit have not been rigorously explored, except under conditions of injury where, for the most part, only single components or tissue samples have been studied. Whereas maintenance or timely re-establishment of flow reduces tissue and neuron injury in both humans and animal models, protection of neuron function in humans has not prevented the evolution of injury despite the inherent mechanisms of neurovascular coupling. However, occlusion of flow to the brain rapidly identifies regions of neuron-vascular vulnerability within the vascular territory-at-risk. These coalesce to become the mature ischaemic lesion. The failure, so far, of clinical trials of neuron protectant agents to achieve detectable tissue salvage could be explained by the vulnerability (and lack of protection) of essential components of the 'unit'. This presentation summarizes evidence and thoughts on this topic. These support the need to understand component interactions within the neurovascular unit.

Inflammation and the neurovascular unit in the setting of focal cerebral ischemia.

Responses to focal cerebral ischemia by neurons and adjacent microvessels are rapid, simultaneous, and topographically related. Recent observations indicate the simultaneous appearance of proteases by components of nearby microvessels that are also expressed by neurons in the ischemic territory, implying that the events could be coordinated. The structural relationship of neurons to their microvascular supply, the direct functional participation of glial cells, and the observation of a highly ordered microvessel-neuron response to ischemia suggest that these elements are arranged in and behave in a unitary fashion, the neurovascular unit. Their roles as a unit in the stimulation of cellular inflammation and the generation of inflammatory mediators during focal cerebral ischemia have not been explored yet. However, components of the neurovascular unit both generate and respond to these influences under the conditions of ischemia. Here we briefly explore the potential inter-relationships of the components of the neurovascular unit with respect to their potential roles in ischemia-induced inflammatory responses.

Vascular matrix adhesion and the blood-brain barrier.

The integrity of the cerebral microvasculature depends on the interaction between its component cells and the extracellular matrix, as well as reorganized cell-cell interactions. In the central nervous system, matrix adhesion receptors are expressed in the microvasculature and by neurons and their supporting glial cells. Cells within cerebral microvessels express both the integrin and dystroglycan families of matrix adhesion receptors. However, the functional significance of these receptors is only now being explored. Endothelial cells and astrocytes within cerebral capillaries co-operate to generate and maintain the basal lamina and the unique barrier functions of the endothelium. Integrins and the dystroglycan complex are found on the matrix-proximate faces of both endothelial cells and astrocyte end-feet. Pericytes rest against the basal lamina. In the extravascular compartment, select integrins are expressed on neurons, microglial cells and oligodendroglia. Significant alterations in both cellular adhesion receptors and their matrix ligands occur during focal cerebral ischaemia, which support their functional significance in the normal state. We propose that matrix adhesion receptors are essential for the maintenance of the integrity of the blood-brain permeability barrier and that modulation of these receptors contributes to alterations in the barrier during brain injury.

Examination of several potential mechanisms for the negative outcome in a clinical stroke trial of enlimomab, a murine anti-human intercellular adhesion molecule-1 antibody: a bedside-to-bench study.

BACKGROUND AND PURPOSE: Enlimomab, a murine monoclonal anti-human intercellular adhesion molecule (ICAM)-1 antibody, had a negative outcome in a multicenter acute-stroke trial. We did a bedside-to-bench study in standardized rat stroke models to explore mechanisms for these untoward results. METHODS: After focal brain ischemia in Wistar rats and spontaneously hypertensive rats (SHR), we administered murine anti-rat ICAM-1 antibody (1A29), subclass-matched murine immunoglobulin (IgG1), or vehicle intravenously. To examine whether rat anti-mouse antibodies were generated against the mouse protein and whether these were deleterious, we sensitized Wistar rats with 1A29 or vehicle 7 days before surgery. Infarct volume, tissue myeloperoxidase activity, neutrophil CD11b expression, and microvascular E-selectin, P-selectin, and ICAM-1 expression were examined 48 hours after surgery. Complement activation was serially assessed for 2 hours after a single injection of either 1A29 or vehicle. RESULTS: 1A29 treatment did not significantly reduce infarct size in either strain. 1A29 sensitization augmented infarct size and generated rat anti-mouse antibodies. Although 1A29 inhibited neutrophil trafficking shown by reduction in brain myeloperoxidase activity, circulating neutrophils were activated and displayed CD11b upregulation. Complement was activated in 1A29-sensitized Wistar rats and 1A29-treated SHR. E-selectin (SHR), endothelial P-selectin (Wistar and SHR), and ICAM-1 (SHR) were upregulated in animals treated with 1A29. CONCLUSIONS: Administration to rats of a murine antibody preparation against ICAM-1, 1A29, elicits the production of host antibodies against the protein, activation of circulating neutrophils, complement activation, and sustained microvascular activation. These observations provide several possible mechanisms for central nervous system-related clinical deterioration that occurred when Enlimomab was given in acute ischemic stroke.

Rapid loss of microvascular integrin expression during focal brain ischemia reflects neuron injury.

The integrity of cerebral microvessels requires the close apposition of the endothelium to the astrocyte endfeet. Integrins alpha1beta1 and alpha6beta4 are cellular matrix receptors that may contribute to cerebral microvascular integrity. It has been hypothesized that focal ischemia alters integrin expression in a characteristic time-dependent manner consistent with neuron injury. The effects of middle cerebral artery occlusion (MCAO) and various periods of reperfusion on microvasclar integrin alpha1beta1 and alpha6beta4 expression were examined in the basal ganglia of 17 primates. Integrin subunits alpha1 and beta1 colocalized with the endothelial cell antigen CD31 in nonischemic microvessels and with glial fibrillary acidic protein on astrocyte fibers. Rapid, simultaneous, and significant disappearance of both integrin alpha1 and beta1 subunits and integrin alpha6beta4 occurred by 2 hours MCAO, which was greatest in the region of neuron injury (ischemic core, Ic), and progressively less in the peripheral (Ip) and nonischemic regions (N). Transcription of subunit beta1 mRNA on microvessels increased significantly in the Ic/Ip border and in multiple circular subregions within Ic. Microvascular integrin alpha1beta1 and integrin alpha6beta4 expression are rapidly and coordinately lost in Ic after MCAO. With loss of integrin alpha1beta1, multiple regions of microvascular beta1 mRNA up-regulation within Ic suggest that microvessel responses to focal ischemia are dynamic, and that multiple cores, not a single core, are generated. These changes imply that microvascular integrity is modified in a heterogeneous, but ordered pattern.

Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: During focal cerebral ischemia, the microvascular matrix (ECM), which participates in microvascular integrity, is degraded and lost when neurons are injured. Loss of microvascular basal lamina antigens coincides with rapid expression of select matrix metalloproteinases (MMPs). Plasminogen activators (PAs) may also play a role in ECM degradation by the generation of plasmin or by MMP activation. METHODS: The endogenous expressions of tissue-type plasminogen activator (tPA), urokinase (uPA), and PA inhibitor-1 (PAI-1) were quantified in 10-microm frozen sections from ischemic and matched nonischemic basal ganglia and in the plasma of 34 male healthy nonhuman primates before and after middle cerebral artery occlusion (MCA:O). RESULTS: Within the ischemic basal ganglia, tissue uPA activity and antigen increased significantly within 1 hour after MCA:O (2P<0.005). tPA activity transiently decreased 2 hours after MCA:O (2P=0.01) in concert with an increase in PAI-1 antigen (2P=0.001) but otherwise did not change. The transient decrease in free tPA antigen was marked by an increase in the tPA-PAI-1 complex (2P<0.001). No significant relations to neuronal injury or intracerebral hemorrhage were discerned. CONCLUSIONS: The rapid increase in endogenous PA activity is mainly due to significant increases in uPA, but not tPA, within the ischemic basal ganglia after MCA:O. This increase and an increase in PAI-1 coincided with latent MMP-2 generation and microvascular ECM degeneration but not neuronal injury.

Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94.

It has been shown recently that matrix metalloproteinases (MMPs) are elevated after cerebral ischemia. In the current study, we investigated the pathophysiologic role for MMP-9 (gelatinase B, EC.3.4.24.35) in a mouse model of permanent focal cerebral ischemia, using a combination of genetic and pharmacologic approaches. Zymography and Western blot analysis demonstrated that MMP-9 protein levels were rapidly up-regulated in brain after ischemic onset. Reverse transcription polymerase chain reaction showed increased transcription of MMP-9. There were no differences in systemic hemodynamic parameters and gross cerebrovascular anatomy between wild type mice and mutant mice with a targeted knockout of the MMP-9 gene. After induction of focal ischemia, similar reductions in cerebral blood flow were obtained. In the MMP-9 knockout mice, ischemic lesion volumes were significantly reduced compared with wild type littermates in male and female mice. In normal wild type mice, the broad spectrum MMP inhibitor BB-94 (batimastat) also significantly reduced ischemic lesion size. However, BB-94 had no detectable protective effect when administered to MMP-9 knockout mice subjected to focal cerebral ischemia. These data demonstrate that MMP-9 plays a deleterious role in the development of brain injury after focal ischemia.

Antithrombotic treatments in acute ischemic stroke.

Ischemic stroke results most commonly from cerebral arterial thrombosis. Antithrombotic agents can reduce the incidence of cerebral embolic events or the extent of tissue injury and neurological outcome. The antiplatelet agents aspirin, ticlopidine, and the combination of dipyridamole and aspirin are associated with a significant reduction in second focal cerebral ischemic events. Oral anticoagulants have a role to reduce the incidence of cardiogenic emboli in patients with mechanical cardiac valves or nonvalvular atrial fibrillation. Both antithrombotics are untested in the acute setting. The recombinant tissue plasminogen activator rt-PA has been shown to significantly increase the number of stroke patients with no or minimal deficit when treated within 3 hours of symptom onset. Additional studies of this and other plasminogen activators by both intravenous and intra-arterial delivery have highlighted limitations to this approach, but also support its role in acute intervention. The risk of intracerebral hemorrhage attends the use of all antithrombotic agents, most notably plasminogen activators. Strategies to decrease this risk are likely to add to beneficial outcome.

Integrin alpha(IIb)beta(3) inhibitor preserves microvascular patency in experimental acute focal cerebral ischemia.

BACKGROUND AND PURPOSE: Platelets become activated and accumulate in brain microvessels of the ischemic microvascular bed after experimental focal cerebral ischemia. The binding of glycoprotein IIb/IIIa (integrin alpha(IIb)beta(3)) on platelets to fibrinogen is the terminal step in platelet adhesion and aggregation. This study tests the hypothesis that inhibition of platelet-fibrin(ogen) interactions may prevent microvascular occlusion after experimental middle cerebral artery occlusion (MCA:O). METHODS: TP9201 is a novel Arg-Gly-Asp (RGD)-containing integrin alpha(IIb)beta(3) inhibitor. Microvascular patency after 3-hour MCA:O and 1-hour reperfusion within the ischemic and nonischemic basal ganglia was compared in adolescent male baboons who received high-dose TP9201 (group A: IC(80) in heparin, n=4), low-dose TP9201 (group B: IC(30) in heparin, n=4), or no treatment (group C: n=4) before MCA:O. RESULTS: After MCA:O, microvascular patency decreased significantly in group C. However, in the ischemic zones of groups A and B compared with group C, patencies were significantly greater in the 4.0- to 7. 5-microm-diameter (capillary) and 7.5- to 30.0-microm-diameter vessels (2P<0.05). A dose-dependent increase in hemorrhagic transformation was seen in group A (3 of 4 animals) compared with group B (1 of 4 animals), and no hemorrhage was visible in group C (chi(2) analysis for trend, P<0.05). CONCLUSIONS: Platelet activation contributes significantly to ischemic microvascular occlusion. Occlusion formation may be prevented by this RGD-integrin alpha(IIb)beta(3) inhibitor at a dose that does not produce clinically significant parenchymal hemorrhage. The effect of microvascular patency on neuron recovery can now be tested.

Advances in the vascular pathophysiology of ischemic stroke.

The cerebral vascular supply is constructed to protect the cerebral hemispheres and brainstem from the consequences of blood flow cessation. Reversal of blood flow around local obstructions is a feature of the microvascular beds of the striatum and cerebral cortex. Cerebral capillaries of these beds consist of endothelial cells, basal lamina, and astrocyte end-feet that sit in close apposition. The interaction of astrocytes with neurons indicates the close relationship of microvessels to neurons. These relationships are altered when blood flow ceases in the supplying artery. Increased endothelial cell permeability and endocytoses lead to edema formation, and matrix degradation is associated with hemorrhage. Autoregulation is lost. Ischemia initiates leukocyte adhesion receptor expression, which is promoted by cytokine generation from the neuropil and activated monocytes. "Preactivation" may further augment the inflammatory responses to ischemia. The activation of cerebral microvessels by ischemia is heterogeneous, involving alterations in integrin-matrix interactions, leukocyte-endothelial cell adhesion, permeability changes, and the "no-reflow" phenomenon due to platelet activation, fibrin formation, and leukocyte adhesion. Ischemia produces swelling of the microvascular endothelium, and rapid detachment and swelling of the astrocyte end-feet. Ischemic injury targets the microvasculature, where the inflammatory responses are initiated and contribute to tissue injury.

Antithrombotic treatments in acute ischemic stroke.

A therapeutic role for antiplatelet agents and anticoagulants within 6 hours of the onset of ischemic stroke symptoms has not been tested. With one exception, their use in early stroke (< 48 hours) did not produce a favorable outcome. The use of rt-PA in appropriate patients presenting with ischemic stroke within 3 hours of symptom onset has been accompanied by significant benefit, which has exceeded the risk of intracerebral hemorrhage in one trial. The recent group of clinical studies has provided evidence for factors which may contribute to hemorrhagic transformation. These studies also demonstrate the following: i) recanalization of carotid and vertebrobasilar artery territory occlusions is technically feasible within 3 to 6 hours of symptom onset, ii) the frequency of hemorrhage is increased in ischemic stroke patients receiving PAs, iii) the interval from symptom onset to treatment to achieve clinical improvement varies individually and contributes to hemorrhagic risk, and iv) the optimal PAs, their dose-rate, and delivery systems have not yet been defined in either the carotid or vertebrobasilar territory. Taken together, the NINDS trial, ECASS, and ECASS II indicate the enormous importance of patient selection to reduce the hemorrhagic risk which accompanies the use of PAs in stroke. However, it is currently not possible to separate benefit from hemorrhagic risk in a given patient based upon simple clinical criteria, although contributors to this risk have been identified. Clearly, attempts to reduce the risk of hemorrhage should contribute to the overall benefit of selected ischemic stroke patients treated with rt-PA and with other PAs. This experience may also provide a clinical basis for the prospective study of antiplatelet agents and anticoagulants in acute ischemic stroke.

Hemorrhagic transformation within 36 hours of a cerebral infarct: relationships with early clinical deterioration and 3-month outcome in the European Cooperative Acute Stroke Study I (ECASS I) cohort.

BACKGROUND AND PURPOSE: The clinical correlates of the varying degrees of early hemorrhagic transformation of a cerebral infarct are unclear. We investigated the cohort of a randomized trial of thrombolysis to assess the early and late clinical course associated with different subtypes of hemorrhagic infarction (HI) and parenchymal hematoma (PH) detected within the first 36 hours of an ischemic stroke. METHODS: We exploited the database of the European Cooperative Acute Stroke Study I (ECASS I), a randomized, placebo-controlled, phase III trial of intravenous recombinant tissue plasminogen activator in acute ischemic stroke. Findings on 24- to 36- hour CT were classified into 5 categories: no hemorrhagic transformation, HI types 1 and 2, and PH types 1 and 2. We assessed the risk of concomitant neurological deterioration and of 3-month death and disability associated with subtypes of hemorrhagic transformation, as opposed to no bleeding. Risks were adjusted for age and extent of ischemic damage on baseline CT. RESULTS: Compared with absence of hemorrhagic transformation, HI1, HI2, and PH1 did not modify the risk of early neurological deterioration, death, and disability, whereas, in both the placebo and the recombinant tissue plasminogen activator groups, PH2 had a devastating impact on early neurological course (odds ratio for deterioration, 32.3; 95% CI, 13. 4 to 77.7), and on 3-month death (odds ratio, 18.0; 95% CI, 8.05 to 40.1). Risk of disability was also higher, but not significantly, after PH2. CONCLUSIONS: Risk of early neurological deterioration and of 3-month death was severely increased after PH2, indicating that large hematoma is the only type of hemorrhagic transformation that may alter the clinical course of ischemic stroke.

Activated microvessels express vascular endothelial growth factor and integrin alpha(v)beta3 during focal cerebral ischemia.

Both vascular endothelial growth factor (VEGF) and integrin alpha(v)beta3 play roles in angiogenesis. In noncerebral vascular systems, VEGF can induce endothelial integrin alpha(v)beta3 expression. However, it is unknown whether VEGF, like integrin alpha(v)beta3, appears in the initial response of microvessels to focal brain ischemia. Their coordinate expression in microvessels of the basal ganglia after middle cerebral artery occlusion (MCAO) in the nonhuman primate model was examined quantitatively. Cells incorporating deoxyuridine triphosphate (dUTP+) by the polymerase I reaction at 1 hour (n = 3), 2 hours (n = 3), and 7 days (n = 4) after MCAO defined the ischemic core (Ic) and peripheral regions. Both VEGF and integrin alpha(v)beta3 were expressed by activated noncapillary (7.5- to 30.0-microm diameter) microvessels in the Ic region at 1 and 2 hours after MCAO. At 7 days after MCAO, the number of VEGF+, integrin alpha(v)beta3+, or proliferating cell nuclear antigen-positive microvessels had decreased within the Ic region. The expressions of VEGF, integrin alpha(v)beta3, and proliferating cell nuclear antigen were highly correlated on the same microvessels using hierarchical log-linear statistical models. Also, VEGF and subunit alpha(v) messenger ribonucleic acids were coexpressed on selected microvessels. Here, noncapillary microvessels are activated specifically early during a focal cerebral ischemic insult and rapidly express VEGF and integrin alpha(v)beta3 together.