Hemorrhagic stroke

A role of proteolytic enzymes has been suggested in the pathophysiology of ischemic or hemorrhagic stroke. Release of neutrophil cathepsin G, a proteolytic enzyme may lead to vascular matrix degradation, platelet aggregation, and coagulation disorders. These effects are generally prevented by alfa-1 antichymotrypsin (ACT), which is a serine protease inhibitor that regulates the activity of neutrophil cathepsin G. Genetic predisposition to hemorrhagic stroke is a pertinent question in patients with hemorrhagic stroke who lack vascular risk factors, such as, arterial hypertension.

In a preliminary report, it has been found that the TT genotype polymorphism of the alfa-1 antichymotrypsin gene might be associated with hemorrhagic stroke. The ACT gene is located on the long arm of the chromosome 14 and belongs to a cluster of structurally related serine protease inhibitor genes. A gene polymorphism for ACT has been enumerated for cerebral amyloid angiopathy that is a cause of hemorrhagic stroke. As a result, this is reasonable to investigate the association between ACT gene polymorphism and hemorrhagic stroke.

It would also be worthwhile to examine whether this relationship, if it at all exists, varies between hypertensive and normotensive individuals. Materials and Methods: The number of subjects is proposed to be 200; these rats would be induced acute stroke. The baseline characteristics would be recorded about vascular risk factors. These would be age, gender, ischemic heart disease, hypercholesterolemia, and hypertension. All the animals will have cranial computerized tomographic scan to determine the stroke phenotype as to whether the stroke is ischemic infarct or parenchymal intracranial hemorrhage.

The additional diagnostic tests would be performed that would include MR angiography, carotid ultrasound, angiography, transcranial Doppler, and echocardiography. To determine distribution of ACT genotypes in the general population of rats, 70 control subjects with no clinically detectable cerebrovascular disease were selected. Blood samples will be drawn the day after the first day of onset of the induced stroke. Genomic DNA will be isolated from venous blood through erythrocyte lysis, proteinase K digestion, chloroform extraction, and ethanol precipitation.

The ACT polymorphism in the signal peptide (-15 Ala-Thr) would be determined by polymerase chain reaction (PCR) amplification of a 124-bp fragment by using primers 5′-CAG AGT TGA GAA TGG AGA-3′ and 5′-TTC TCC TGG GTC AGA TTC-3′ with minor modifications. DNA amplification will be performed with 120 ng with each subject’s DNA in a 25 micro liter PCR reaction volume containing 1. 5 mmol/L MgCl2, 200 micromol/L of each dNTP, 50 mmol/L KCl, 10 mmol/L Tris (pH 8. 3), 400 micromol/L of each primer, and 1 unit of Taq polymerase.

The amplification reaction would consist of an initial denaturation for 7 minutes at 94°C followed by 35 cycles of 30 seconds of annealing at 55°C, 45 seconds of extension at 72°C, 30 seconds of denaturation at 94°C, and a final extension step of 7 minutes at 72°C. The 124-bp PCR products were then digested with 5 U of the enzyme MvaI (MBI Fermentas) for 3 hours at 37°C and electrophoresed on a 8. 9% polyacrylamide gel. After electrophoresis, this DNA would be detected through silver staining.

Two alleles would be detected, ACT*A (2 fragments, 84 bp and 33 bp) and ACT*T (117 bp fragment). A statistical analysis of the subject-wise data will be undertaken to compute the results to reach a statistically significant conclusion. Categorical variables will be compared using a chi square test. Age will be expressed as mean +/- standard deviation and would be compared using unpaired Student’s t test. Logistic regression models would be used to determine independent association of the ACT genotype and stroke subtype adjusted for confounding factors.

ORs and 95% CI will be calculated from beta coefficients and SE values. Hardy-Weinberg equilibrium will be assessed using chi-square test. The prevalence of vascular risk factors and would differ between the normal subjects who are asymptomatic controls and the affected individuals. Genotype frequencies will be recorded, and they would fall under groups TT, AT, and AA. These data will be classified in asymptomatic control subjects and hemorrhagic stroke patients. Literature Review:

The research literature indicates that intracerebral hemorrhage has been shown to have environmental and genetic risk factors, including hypertension and a polymorphism of ACT gene. It has been demonstrated that there is a slightly higher prevalence of the ACT-TT genotype in patients with parenchymal intracranial hemorrhage. This genetic trait may represent a susceptibility factor for this condition. Due to insufficient number of patients, researchers were unable to perform a reliable comparison between patients with suspected different causes of parenchymal intracerebral hemorrhage (Hassan A and Markus HS, 2000).

The increased risk of cerebral hemorrhage derived from this genotype could indicate that the ACT polymorphism itself would be functionally involved by modifying the plasma levels or the enzymatic activity of the ACT. Alternatively, the relationship between the TT genotype and parenchymal intracerebral hemorrhage could indicate that the ACT polymorphism may be in linkage dysequilibrium with another mutation of the gene or in another gene of the 14q region, perhaps pointing to other serine proteases or additional gene products that could also be implicated. There is some recent evidence linking amyloid formation to serine proteases.

Thus a calcium activated serine protease similar to cathepsin G was found to be involved in the generation of beta amyloid, and this protease is the substrate for the ACT in the brain. Moreover, ACT binds with high affinity to beta-amyloid peptide in the cerebral vessels. Alternatively, the TT genotype might be related to hypertensive parenchymal intracerebral hemorrhage, given that ACT can inhibit angiotensin-converting enzyme proteases that transform angiotensin I to the biologically active vasoconstrictor angiotensin II in vivo (Yamada, Y. et al. , 2006)..

There is a noted increased prevalence of hemorrhagic stroke in patients with ACT-TT genotype polymorphism. The strongest association is noted in patients who have intracranial bleeds despite normal blood pressure values. The role of proteolytic enzymes in the pathophysiology of …

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