Gregory S. Makowski

Binding of Latent Matrix Metalloproteinase 9 to Fibrin: Activation via a Plasmin-Dependent Pathway

The binding of two matrix metalloproteinases (MMP) to fibrin was evaluated. MMP-2 (72-kDa) and MMP-9 (92-, 130-, and 225-kDa) were selected since both contain a fibronectin-like region and fibronectin binds fibrin. Gelatin zymography indicated selective and dose dependent binding of MMP-9 to fibrin. No MMP-2 binding to fibrin occurred. Densitometry revealed that the 130- and 225-kDa forms demonstrated similar sigmoidal binding profiles whereas 92-kDa uptake was hyperbolic. Fibronectin and TIMP-1 competition studies indicated that the fibronectin and C-terminal MMP-9 domains, respectively, were not involved with fibrin binding. The MMP-9 collagen-like region may be of regulatory significance since type I and II fibrillar and type IV basement membrane collagens demonstrated fibrin binding. During fibrinolysis, latent fibrin-bound MMP-9 was processed to lower molecular weight forms consistent with proteolytic activation. This process was inhibited by ∈-aminocaproic acid, indicating a plasmin-dependent pathway. The significance of these findings to procoagulant activity and MMP-mediated extracellular matrix destruction during inflammation and tumor invasion and metastasis is discussed.

Identification and partial characterization of three calcium- and zinc-independent gelatinases constitutively present in human circulation

Three constitutive gelatinases in human plasma were identified and characterized relative to known matrix metalloproteinase (MMP) gelatinases: MMP‐2 (fibroblast 72‐kDa) and MMP‐9 (neutrophil 92‐, 130‐, and 225‐kDa). Substrate gel electrophoresis (gelatin zymography) revealed an apparent Mw of 78‐, 82‐, and 89‐ kDa for these gelatinases. Densitometry revealed that MMP‐9 and MMP‐2 were highly calcium sensitive requiring 50‐150 μM and 500 μM calcium for half‐maximal activity, respectively. Of the new gelatinases, only the 89‐kDa form demonstrated slight calcium activation. The three gelatinases were unaffected by known MMP inhibitors: EDTA (5 mM), 1,10‐phenanthroline (2 mM), and pepstatin (18 μM). Serine and thiol protease inhibitors (leupeptin, aprotinin, PMSF, TLCK, TPCK, antichymostatin, antipain) were also ineffective. Solution‐phase IEF revealed that the 78‐ and 82‐kDa forms focused at neutral pl 6.72‐7.95 whereas the 89‐kDa focused at an acidic pI 4.89‐5.18 (similar to neutrophil and fibroblast forms). The data indicate that these gelatinases are not MMPs or partially activated MMPs. Their role in normal and pathological conditions is not known.

The interactions of zinc, nickel, and cadmium with Xenopus transcription factor IIIA, assessed by equilibrium dialysis

Transcription factor IIIA (TFIIIA) was isolated from Xenopus ovary and treated with 1,10-phenanthroline to remove zinc. The interactions of apoTFIIIA with Zn2+, Ni2+, and Cd2+ were studied by equilibrium dialysis under anaerobic conditions (pH 7.0, 25 degrees C), using 65ZnCl2, 63NiCl2, and 109CdCl2 as the radioligands. The data for binding of Zn2+, Ni2+, and Cd2+ to apoTFIIIA were best-fitted by a model with two classes of binding sites. For Zn2+, the apparent dissociation constants (KdlZn and Kd2Zn) for the high- and low-affinity sites were 1.0 x 10(-8) and 2.6 x 10(-5) M; the apparent binding capacities of the two classes were 0.8 +/- 0.5 and 9.6 +/- 0.3 g-atoms of Zn/mol; the Hill coefficient was 1.18, consistent with positive cooperativity of Zn-binding sites. For Ni2+, the apparent KdlNi and Kd2Ni values were 2.3 x 10(-5) and 5.2 x 10(-4) M; the apparent binding capacities were 2.3 +/- 0.6 and 8.6 +/- 0.6 g-atoms of Ni/mol; the Hill coefficient was 1.20, consistent with positive cooperativity of Ni-binding sites. For Cd2+, the apparent KdlCd and Kd2Cd values were 2.8 x 10(-6) and 1.6 x 10(-4) M; the apparent binding capacities were 0.9 +/- 0.3 and 2.4 +/- 0.5 g-atoms of Cd/mol; the Hill coefficient was 0.53, consistent with negative cooperativity or heterogeneity of Cd-binding sites. This study has the following significance: First, it helps to resolve a controversy about the zinc content of purified TFIIIA. Second, it shows that the KdlZn of apoTFIIIA is less than the reported KdZn of thionein, consistent with the hypothesis that thionein modulates gene expression by competing with TFIIIA and other Zn-finger proteins for intracellular Zn2+ stores. Third, it confirms previous indirect evidence that the affinity of apoTFIIIA for Zn2+ is much greater than for Cd2+, and that the affinity for Cd2+ is greater than for Ni2+.

Amorphous Calcium Phosphate-Mediated Binding of Matrix Metalloproteinase-9 to Fibrin is Inhibited by Pyrophosphate and Bisphosphonate

Coordinate regulation of fibrinolytic and collagenolytic systems is essential for normal tissue remodeling and wound healing. To define the molecular mechanisms which link these two proteolytic systems, we have investigated the role of fibrin in matrix metalloproteinase (MMP) function. Both active and latent forms of MMP-9 (gelatinase B) bind to fibrin in a selective, dose-dependent manner; latent enzyme is activated by plasmin during fibrinolysis. Fibrin binding of MMP-9 is mediated by amorphous calcium phosphate (ACP), and proceeds in a step-wise fashion with formation of ACP as the first and rate-limiting step. MMP-9 rapidly binds preformed ACP to yield a transient ACP:MMP-9 complex that avidly binds fibrin. Here we report the effect(s) on fibrin:ACP:MMP-9 formation/dissociation of pyrophosphate (POP), an endogenous calcification inhibitor, and its bisphosphonate analog, alendronate (PCP). MMP-9 was obtained from neutrophil lysate and ACP formation was monitored turbidimetrically. Free MMP-9, ACP:MMP-9 and fibrin:ACP:MMP-9 complexes were analyzed by gelatin zymography. POP at physiologic concentrations (0.5−2.5 μM) inhibited both ACP formation and subsequent fibrin binding of MMP-9 at orthophosphate concentrations of 250 μM. PCP exhibited a similar inhibitory effect. With both substances, inhibition was slightly overcome (>2.5 μM) by higher phosphate (500 μM). In contrast, supraphysiologic concentrations of either POP or PCP (>50 μM) were required to inhibit MMP-9 binding to preformed ACP or to induce dissociation of preformed ACP:MMP-9 complexes (50–100 μM). Neither POP nor PCP had any effect on preformed fibrin:ACP:MMP-9 at concentrations up 1 mM. POP is an endogenous by-product of numerous metabolic pathways and may regulate bone turnover, soft tissue calcification, and contribute to the pathogenesis of calcium pyrophosphate crystal disease (CPPD). These studies support another role for POP and fibrin:ACP:MMP-9 complexes in physiologic and pathologic processes, including tumorigenesis and cancer metastasis.

AMPLIFICATION OF GUTHRIE CARD DNA : EFFECT OF GUANIDINE THIOCYANATE ON BINDING OF NATURAL WHOLE BLOOD PCR INHIBITORS

Amplification of DNA from whole blood collected on Guthrie card filter paper presents considerable technical obstacles due to the presence of natural PCR inhibitors (protein, heavy metals, heme, and heme degradation products) and low copy number of genomic material. For this purpose we evaluated guanidine thiocyanate‐impregnated filter paper (GT‐903), a DNA collection device designed specifically to bind PCR inhibitors and preserve DNA in an aqueous extractable form. Compared to standard 903, which retains DNA and elutes inhibitors during aqueous extraction, we found GT‐903 retained 90% of protein, hemoglobin, and iron. SDS‐PAGE analysis indicated that the majority of the protein released from standard 903 corresponded to albumin (70‐) and globin (15‐kDa); negligible levels of these proteins were eluted from GT‐903. To evaluate PCR efficiency, we amplified the 491 bp region encoding the cystic fibrosis ΔF508 mutation. Using comparable template, we found GT‐903 amplification more efficient than standard 903 following qualitative (TBE‐PAGE) and quantitative (anti‐dsDNA EIA) determination. We conclude that GT‐903 provides a good DNA collection device and addresses the complications associated with natural PCR inhibitors. J. Clin. Lab. Anal. 11:87–93.

Binding of Matrix Metalloproteinase 9 to Fibrin is Mediated by Amorphous Calcium-Phosphate

In our previous study we demonstrated selective, dose-dependent binding of matrix metalloproteinase-9 (MMP-9), a neutrophil collagenase, to fibrin. Here we investigated the mechanism of this interaction. We found that MMP-9 to fibrin was dependent on formation of a calcium-phosphate intermediate. The intermediate was precipitable by centrifugation and contained a Ca/P ratio of 1.52–1.54, consistent with amorphous calcium-phosphate (ACP). ACP formation exhibited a temperature optimum at 37°C. Gelatin zymography revealed that interaction of ACP with MMP-9 resulted in formation of a high molecular weight ACP : MMP-9 complex which was required for MMP-9 binding to fibrin. Complex formation was dependent on the generation of viable ACP that required both calcium (7.5–10 mM) and phosphate (225–250 μM) (Ca × P product range, 1.7–2.5 mM2). Carbonate (CO3) and sulfate (SO4) were ineffective as calcium counteranions. Preformed ACP rapidly complexed MMP-9. Thus ACP formation was rate-limiting for MMP-9 fibrin binding activity. No MMP-9 fibrin binding activity was noted at 25°C, an observation consistent with lack of ACP production. The significance of these findings is discussed with respect to normal and pathologic wound healing.

Interaction of amorphous calcium phosphate with fibrin in vitro causes decreased fibrinolysis and altered protease profiles: implications for atherosclerotic disease.

Previously, we demonstrated that amorphous calcium phosphate (ACP), chemical precursor to apatite, strongly interacted with fibrin and facilitated binding of matrix metalloproteinase (MMP)-9, a type IV collagenase. Plasmin-dependent fibrinolysis resulted in coordinate MMP-9 activation. Here we report on the effect(s) of ACP on fibrin degradation and binding of endogenous plasma proteases. Electrophoresis (8.5% SDS-PAGE) revealed that fibrin formed in the presence of ACP demonstrated characteristic γ-γ dimers (90-kDa) and β-monomers (55-kDa), but resisted spontaneous fibrinolysis (72 h, 37°C) or degradation by plasminogen activators (uPA, tPA). Casein zymography revealed an ACP-dependent decrease in fibrin binding of a low molecular weight (Mw) protease triplet (47-, 43-, 42-kDa) and increased fibrin binding of two high Mw proteases (94- and 84-kDa). The low Mw triplet also possessed gelatinolytic activity, but was not an MMP since 1,10-phenanthroline was ineffective as an inhibitor. Fibrin-binding proteases were inhibited to some degree by the serine protease inhibitor aprotinin. Competition/dissociation experiments with ∈-aminocaproic acid revealed that the low Mw triplet lacked kringle regions whereas the 94- and 84-kDa proteases were tentatively identified and glu-/lys-plasmin(ogen)s. The triplet may, however, represent one or more kringle deficient mini-plasminogen(s), since electrophoretic mobility and substrate specificity was similar to elastase-generated mini-plasminogen. To explore these findings in a clinically relevant setting, a series of plasma samples was collected from a patient with unstable angina prior to, during, and post coronary artery bypass graft (CABG) surgery. Fibrin formed from plasma collected during and immediately post CABG was associated with increased fibrinolytic capacity and enhanced binding of a) MMP-9, b) the low Mw protease triplet (described above), and c) PA (as putative 110-kDa tPA:PAI-1 complex). The relevance of these findings to pathologic calcification of atherosclerotic plaques is discussed.

Practical interpretation of CYP2D6 haplotypes: Comparison and integration of automated and expert calling.

We describe a population genetic approach to compare samples interpreted with expert calling (EC) versus automated calling (AC) for CYP2D6 haplotyping. The analysis represents 4812 haplotype calls based on signal data generated by the Luminex xMap analyzers from 2406 patients referred to a high-complexity molecular diagnostics laboratory for CYP450 testing. DNA was extracted from buccal swabs. We compared the results of expert calls (EC) and automated calls (AC) with regard to haplotype number and frequency. The ratio of EC to AC was 1:3. Haplotype frequencies from EC and AC samples were convergent across haplotypes, and their distribution was not statistically different between the groups. Most duplications required EC, as only expansions with homozygous or hemizygous haplotypes could be automatedly called. High-complexity laboratories can offer equivalent interpretation to automated calling for non-expanded CYP2D6 loci, and superior interpretation for duplications. We have validated scientific expert calling specified by scoring rules as standard operating procedure integrated with an automated calling algorithm. The integration of EC with AC is a practical strategy for CYP2D6 clinical haplotyping.

State-wide hospital clinical laboratory plan for measuring cholinesterase activity for individuals suspected of exposure to nerve agent chemical weapons.

Background. Hospital laboratories currently lack the capacity to provide emergency determination of cholinesterase activity. Methods. We have developed a hospital-based 3-tiered system to test plasma for butyrylcholinesterase (BChE) activity and whole blood for red cell acetylcholinesterase (AChE) activity using available technology and personnel. Interagency communications, toxidrome definition, and patient triage will be coordinated by the Connecticut Department of Public Health and the Poison Control Center. Data. Initial BChE data documents good precision between institutions (coefficient of variation < 8%). Summary. Laboratory testing of plasma or blood for cholinesterase activity is important in the management of nerve agent exposure and in ruling out disease in those with non-specific symptoms in the setting of a terrorist attack or accidental exposure. Rapid availability of strong hospital-based analytic support in a smoothly functioning network of clinical, public health, and laboratory services will facilitate overall regional response to chemical terrorism or large scale HazMat events.

Gelatinolytic and fibrinolytic activity in fresh-frozen plasma

Fresh-frozen plasma (FFP) was evaluated for gelatinolytic and fibrinolytic activity. Gelatin zymography revealed that gelatinase A (MMP-2) was constitutively present in FFP whereas gelatinase B (MMP-9) was present at variable levels. The presence of MMP-9 likely represents differential release from neutrophils during FFP collection or processing. Although fibrin matrices generated from FFP or freshly prepared plasma contained characteristic crosslinked gamma-gamma dimers and beta-monomers, matrices generated from FFP were resistant to spontaneous plasmin-dependent fibrinolysis. This observation likely stems from the plasminogen activator instability and could potentially lead to a hypofibrinolytic state. The impact of these in vitro findings to protease balance in patients receiving multiple FFP doses remains to be determined.